Antigen associated with rheumatoid arthritis

ABSTRACT

The invention relates to a binding member that binds the Extra Domain-A (ED-A) isoform of fibronectin for the detection and treatment of rheumatoid arthritis.

CROSS REFERENCE TO RELATED APPLICATIONS

This is the U.S. National Stage of International Application No.PCT/EP2008/009070, filed Oct. 27, 2008, which was published in Englishunder PCT Article 21(2), which in turn claims the benefit of U.S.Provisional Application No. 60/983,606, filed Oct. 30, 2007, which isincorporated herein in its entirety.

JOINT RESEARCH AGREEMENT

This application describes and claims invention(s) that were developedunder a written joint research agreement between Philogen S.p.A. andPhilochem AG, that was in effect on or before the date the invention(s)were made.

The present invention relates to the detection and treatment ofrheumatoid arthritis (RA). The invention involves use of a bindingmember that binds the ED-A isoform of fibronectin, especially a bindingmember that binds domain ED-A of fibronectin.

Rheumatoid arthritis (RA) is a chronic inflammatory and destructivejoint disease that affects 0.5-1% of the population in theindustrialized world and commonly leads to significant disability and aconsequent reduction in quality of life.

Angiogenesis in the synovial membrane of patients with RA is consideredto be an important early step in pathogenesis and in the perpetuation ofdisease (Taylor, 2002). As in neoplastic disease, angiogenesis feeds theexpanding synovium (Walsh et al., 1998). Blood vessel growth probablycontributes to the proliferation of the inflammatory synovial pannus aswell as to the ingress of inflammatory leukocytes into the synovialtissue. Synovium of patients with RA contained increased amounts offibroblast growth factor-2 (FGF-2) and of vascular endothelial growthfactor (VEGF) (Koch, 2003). Serum VEGF concentrations correlate withdisease activity and fall, when synovitis is successfully suppressed bytherapy (Taylor, 2002).

Fibronectin (FN) is a glycoprotein and is widely expressed in a varietyof normal tissues and body fluids. It is a component of theextracellular matrix (ECM), and plays a role in many biologicalprocesses, including cellular adhesion, cellular migration, haemostasis,thrombosis, wound healing, tissue differentiation and oncogenictransformation.

Different FN isoforms are generated by alternative splicing of threeregions (ED-A, ED-B, IIICS) of the primary transcript FN pre-mRNA, aprocess that is modulated by cytokines and extracellular pH (Balza 1988;Carnemolla 1989; Borsi 1990; Borsi 1995). Fibronectin contains twotype-III globular extra-domains which may undergo alternative splicing:ED-A and ED-B (ffrench-Constant 1995, Hynes 1990, Kaspar et al. 2006).The ED-As of mouse fibronectin and human fibronectin are 96.7% identical(only 3 amino acids differ between the two 90 amino acid sequences, seeFIG. 2).

Expression of the ED-A of fibronectin has been reported in tumour cellsand in solid tumours at the mRNA level in breast cancer (Jacobs et al.2002, Matsumoto et al. 1999) and liver cancer (Oyama et al. 1989, Tavianet al. 1994) and at the level of isolated protein in fibrosarcoma,rhabdomyosarcoma and melanoma (Borsi et al. 1987).

At the immunohistochemical level, the presence of ED-A has been detectedin the extracellular matrix (ECM) of odontogenic tumours (Heikinheimo etal. 1991) and hepatocellular carcinoma (Koukoulis et al. 1995). Incontrast, ED-A has been detected in the stroma of malignant breastneoplasms (Koukoulis et al. 1993), and in the blood vessels and basementmembranes of well-differentiated renal cell carcinoma (Lohi et al.1995). However, in less-differentiated renal cell carcinoma (Lohi et al.1995) and papillary carcinoma of the thyroid (Scarpino et al. 1999) ED-Ahas been detected in the blood vessels, basement membranes and tumourstroma. The presence of ED-A in the vasculature of gliomas has also beenreported (Borsi et al. 1998). Thus, the pattern of ED-A expressionreported for different types of tumours is highly variable.

Antibody-based targeted delivery of bioactive agents to sites ofangiogenesis is an attractive therapeutic strategy for cancer treatment,but is largely unexplored for chronic inflammatory diseases. We havepreviously demonstrated that the ED-B domain of fibronectin, a marker ofangiogenesis, is expressed in psoriatic lesions in patients and in amouse model of psoriasis as well as in arthritic paws in thecollagen-induced mouse model of rheumatoid arthritis. Using bothradioactive and fluorescent techniques, the human monoclonal antibodyL19, specific to EDB, was found to selectively localize at sites ofinflammation in vivo, following intravenous administration. Theseresults suggest a therapeutic potential for the L19-based selectivedelivery of bioactive compounds to sites of inflammation (Trachsel,2007; PCT/EP2007/004044).

It has been shown before by in-situ-hybridisation that other than ED-Balso the ED-A domain of fibronectin can be present in human arthriticspecimens (Berndt et al., 1998; Kriegsmann et al., 2004).

We show herein that anti-EDA antibody, such as the F8 antibody disclosedherein, is able to give a stronger staining pattern on human arthriticspecimens compared with the anti-EDB-antibody L19 and theanti-tenascin-C antibodies F16 and G11.

Furthermore, using both radioactive and fluorescent techniques, thehuman monoclonal antibody F8, specific to ED-A, was found to selectivelylocalize at sites of inflammation in vivo, following intravenousadministration.

Accordingly, ED-A of fibronectin is indicated as a vascular marker ofrheumatoid arthritis.

Binding molecules such as antibody molecules that bind the A-FN and/orthe ED-A of fibronectin represent novel agents which may be used for thepreparation of a medicament for the treatment of rheumatoid arthritis(RA).

This invention provides the use of a binding member, e.g. an antibodymolecule, that binds the Extra Domain-A (ED-A) isoform of fibronectin(A-FN), for the preparation of a medicament for the treatment ofrheumatoid arthritis. The invention also provides the use of a bindingmember, e.g. an antibody molecule, that binds the ED-A of fibronectinfor the preparation of a medicament for the treatment of rheumatoidarthritis.

The invention further provides the use of a binding member, e.g. anantibody molecule, that binds the ED-A isoform of fibronectin fordelivery, to sites of rheumatoid arthritis, of a molecule conjugated tothe binding member. The invention also provides the use of a bindingmember, e.g. an antibody molecule, that binds the ED-A of fibronectinfor delivery, to sites of rheumatoid arthritis, of a molecule conjugatedto the binding member. The binding member may be used for themanufacture of a medicament for delivery of such a molecule.

The invention provides the use of a binding member, e.g. an antibodymolecule, that binds the ED-A isoform of fibronectin for the manufactureof a diagnostic product for use in diagnosing rheumatoid arthritis. Theinvention also provides the use of a binding member, e.g. an antibodymolecule, that binds the ED-A of fibronectin for the manufacture of adiagnostic product for use in diagnosing rheumatoid arthritis.

The invention further provides a method of detecting or diagnosingrheumatoid arthritis in a human or animal comprising:

-   -   (a) administering to the human or animal a binding member, e.g.        an antibody molecule, which binds the ED-A of fibronectin, and    -   (b) determining the presence or absence of the binding member in        sites of rheumatoid arthritis of the human or animal body;        wherein localisation of the binding member to sites of        rheumatoid arthritis indicates the presence of rheumatoid        arthritis.

The present invention provides a method of treating rheumatoid arthritisin an individual comprising administering to the individual atherapeutically effective amount of a medicament comprising a bindingmember, e.g. an antibody molecule, which binds the ED-A isoform offibronectin. The present invention also provides a method of treatingrheumatoid arthritis in an individual comprising administering to theindividual a therapeutically effective amount of a medicament comprisinga binding member, e.g. an antibody molecule, which binds the ED-A offibronectin.

The present invention provides a composition comprising a bindingmember, e.g. an antibody molecule, which binds the ED-A isoform offibronectin, for use in a method of treating rheumatoid arthritis in anindividual comprising administering to the individual a therapeuticallyeffective amount of a medicament comprising a binding member, e.g. anantibody molecule, which binds the ED-A isoform of fibronectin. Thepresent invention also provides a composition comprising a bindingmember, e.g. an antibody molecule, which binds the ED-A of fibronectin,for use in a method of treating rheumatoid arthritis in an individualcomprising administering to the individual a therapeutically effectiveamount of a medicament comprising a binding member, e.g. an antibodymolecule, which binds the ED-A of fibronectin.

The invention provides a method of delivering a molecule to theneovasculature of sites of rheumatoid arthritis in a human or animalcomprising administering to the human or animal a binding member, e.g.an antibody molecule, which binds the ED-A isoform of fibronectin,wherein the binding member is conjugated to the molecule. The inventionalso provides a method of delivering a molecule to the neovasculature ofsites of rheumatoid arthritis in a human or animal comprisingadministering to the human or animal a binding member, e.g. an antibodymolecule which binds the ED-A of fibronectin, wherein the binding memberis conjugated to the molecule.

A binding member for use in the invention may be an antibody which bindsthe ED-A isoform of fibronectin and/or the ED-A of fibronectin,comprising one or more complementarity determining regions (CDRs) ofantibody H1, B2, C5, D5, E5, C8, F8, F1, B7, E8 or G9, or variantsthereof. Preferably, a binding member for use in the invention is anantibody which binds the ED-A isoform of fibronectin and/or the ED-A offibronectin, comprising one or more complementarity determining regions(CDRs) of antibody B2, C5, D5, C8, F8, B7 or G9, or variants thereof.Most preferably, a binding member for use in the invention is anantibody which binds the ED-A isoform of fibronectin and/or the ED-A offibronectin, comprising one or more complementarity determining regions(CDRs) of antibody F8 or variants thereof.

A binding member for use in the invention may comprise a set of H and/orL CDRs of antibody H1, B2, C5, D5, E5, C8, F8, F1, B7, E8 or G9, or aset of H and/or L CDRs of antibody H1, B2, C5, D5, E5, C8, F8, F1, B7,E8 or G9 with ten or fewer, e.g. one, two, three, four, or five, aminoacid substitutions within the disclosed set of H and/or L CDRs.Preferably, a binding member for use in the invention comprises a set ofH and/or L CDRs of antibody B2, C5, D5, C8, F8, B7 or G9 with ten orfewer, e.g. one, two, three, four, or five, amino acid substitutionswithin the disclosed set of H and/or L CDRs. Preferably, a bindingmember for use in the invention comprises a set of H and/or L CDRs ofantibody F8 with ten or fewer, e.g. one, two, three, four, or five,amino acid substitutions within the disclosed set of H and/or L CDRs.

Substitutions may potentially be made at any residue within the set ofCDRs, and may be within CDR1, CDR2 and/or CDR3.

For example, a binding member for use in the invention may comprise oneor more CDRs as described herein, e.g. a CDR3, and optionally also aCDR1 and CDR2 to form a set of CDRs.

A binding member for use in the invention may also comprise an antibodymolecule, e.g. a human antibody molecule. The binding member normallycomprises an antibody VH and/or VL domain. VH domains of binding membersare also provided for use in the invention. Within each of the VH and VLdomains are complementarity determining regions, (“CDRs”), and frameworkregions, (“FRs”). A VH domain comprises a set of HCDRs, and a VL domaincomprises a set of LCDRs. An antibody molecule may comprise an antibodyVH domain comprising a VH CDR1, CDR2 and CDR3 and a framework. It mayalternatively or also comprise an antibody VL domain comprising a VLCDR1, CDR2 and CDR3 and a framework. The VH and VL domains and CDRs ofantibodies H1, B2, C5, D5, E5, C8, F8, F1, B7, E8 and G9 are describedherein. All VH and VL sequences, CDR sequences, sets of CDRs and sets ofHCDRs and sets of LCDRs disclosed herein represent embodiments of abinding member for use in the invention. As described herein, a “set ofCDRs” comprises CDR1, CDR2 and CDR3. Thus, a set of HCDRs refers toHCDR1, HCDR2 and HCDR3, and a set of LCDRs refers to LCDR1, LCDR2 andLCDR3. Unless otherwise stated, a “set of CDRs” includes HCDRs andLCDRs.

A binding member for use in the invention may comprise an antibody VHdomain comprising complementarity determining regions HCDR1, HCDR2 andHCDR3 and a framework, wherein HCDR1 is SEQ ID NO: 3, 23, 33, 43, 53,63, 73, 83, 93, 103 or 113, and wherein optionally HCDR2 is SEQ ID NO: 4and/or HCDR3 is SEQ ID NO: 5. Preferably, the HCDR1 is SEQ ID NO: 23,33, 43, 53, 73, 83 or 103. Most preferably, the HCDR1 is SEQ ID NO: 83.

Typically, a VH domain is paired with a VL domain to provide an antibodyantigen-binding site, although as discussed further below a VH or VLdomain alone may be used to bind antigen. Thus, a binding member for usein the invention may further comprise an antibody VL domain comprisingcomplementarity determining regions LCDR1, LCDR2 and LCDR3 and aframework, wherein LCDR1 is SEQ ID NO: 6, 26, 36, 46, 56, 66, 76, 86,96, 106 or 116 and wherein optionally LCDR2 is SEQ ID NO: 7 and/or LCDR3is SEQ ID NO: 8. Preferably, the LCDR1 is SEQ ID NO: 26, 36, 46, 56, 76,86 or 106. Most preferably, the LCDR1 is SEQ ID NO: 86.

A binding member for use in the invention may be an isolated antibodymolecule for the ED-A of fibronectin, comprising a VH domain and a VLdomain, wherein the VH domain comprises a framework and a set ofcomplementarity determining regions HCDR1, HCDR2 and HCDR3 and whereinthe VL domain comprises complementarity determining regions LCDR1, LCDR2and LCDR3 and a framework, and wherein

-   HCDR1 has amino acid sequence SEQ ID NO: 3, 23, 33, 43, 53, 63, 73,    83, 93, 103 or 113,-   HCDR2 has amino acid sequence SEQ ID NO: 4,-   HCDR3 has amino acid sequence SEQ ID NO: 5,-   LCDR1 has amino acid sequence SEQ ID NO: 6, 26, 36, 46, 56, 66, 76,    86, 96, 106 or 116;-   LCDR2 has amino acid sequence SEQ ID NO: 7; and-   LCDR3 has amino acid sequence SEQ ID NO: 8.

One or more CDRs or a set of CDRs of an antibody may be grafted into aframework (e.g. human framework) to provide an antibody molecule for usein the invention. Framework regions may comprise human germline genesegment sequences. Thus, the framework may be germlined, whereby one ormore residues within the framework are changed to match the residues atthe equivalent position in the most similar human germline framework. Abinding member for use in the invention may be an isolated antibodymolecule having a VH domain comprising a set of HCDRs in a humangermline framework, e.g. DP47. Normally the binding member also has a VLdomain comprising a set of LCDRs, e.g. in a human germline framework.The human germline framework of the VL domain may be DPK22.

A VH domain for use in the invention may have amino acid sequence SEQ IDNO: 1, 21, 31, 41, 51, 61, 71, 81, 91, 101 or 111. Preferably, a VHdomain for use in the invention has amino acid sequence SEQ ID NO: 21,31, 41, 51, 71, 81 or 101. Most preferably, a VH domain for use in theinvention has amino acid sequence SEQ ID NO: 81. A VL domain for use inthe invention may have the amino acid SEQ ID NO: 2, 22, 32, 42, 52, 62,72, 82, 92, 102 or 112. Preferably, a VL domain for use in the inventionhas amino acid SEQ ID NO: 22, 32, 42, 52, 72, 82 or 102. Mostpreferably, a VL domain for use in the invention has amino acid SEQ IDNO: 82.

A binding member for use in the invention may be or comprise a singlechain Fv (scFv), comprising a VH domain and a VL domain joined via apeptide linker. The skilled person may select an appropriate length andsequence of linker, e.g. at least 5 or 10 amino acids in length, up toabout 15, 20 or 25 amino acids in length. The linker may have the aminoacid sequence GSSGG (SEQ ID NO: 28). The scFv may consist of or compriseamino acid sequence SEQ ID NO: 9.

A single chain Fv (scFv) may be comprised within a mini-immunoglobulinor small immunoprotein (SIP), e.g. as described in (Li et al., 1997). Asip may comprise an scFv molecule fused to the CH4 domain of the humanIgE secretory isoform IgE-S2 (ε_(S2)-CH4; Batista et al., 1996) formingan homo-dimeric mini-immunoglobulin antibody molecule.

Alternatively, a binding member for use in the invention may comprise anantigen-binding site within a non-antibody molecule, normally providedby one or more CDRs e.g. a set of CDRs in a non-antibody proteinscaffold. Binding members, including non-antibody and antibodymolecules, are described in more detail elsewhere herein.

A binding member for use in the invention may be conjugated to amolecule that has biocidal, cytotoxic immunosuppressive oranti-inflammatory activity. Interleukin-10 is an advantageous moleculefor conjugation with a binding member in accordance with the presentinvention, and useful in treatment of rheumatoid arthritis. Furthermore,a binding member for use in the invention may be conjugated to aradioisotope, a detectable lable or a photosensitizer.

These and other aspects of the invention are described in further detailbelow.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the results of immunohistochemistry on human arthriticspecimens using antibodies directed to markers of angiogenesis. Darkerstaining indicates strong expression of the antigen, visualized by whitearrows. F8 is an antibody molecule that binds ED-A, disclosed herein,L19 is an antibody molecule that binds ED-B (e.g. Pini et al. 1998), F16and G11 are antibody molecules that bind Tenascin-C domains A1 and C,respectively (WO2006/050834).

FIG. 2 shows the results of immunofluorescence analysis on humanarthritic specimens using the F8 antibody molecule directed against theED-A domain of fibronectin. White staining indicates strong expressionof the antigen.

FIG. 3 shows an alignment between A: the human ED-A (top sequence) andB: the mouse ED-A (bottom sequence). The asterisks indicate the aminoacid positions where the amino acids of the human ED-A and the mouseED-A are identical.

FIG. 4A shows the nucleotide sequence of the anti-ED-A antibody H1 heavychain (VH) (SEQ ID NO: 12). The nucleotide sequence of the heavy chainCDR1 of anti-ED-A antibody H1 is underlined. The nucleotide sequence ofthe heavy chain CDR2 of the anti-ED-A antibody H1 is shown in italicsand underlined. The nucleotide sequence of the heavy chain CDR3 ofanti-ED-A antibody H1 is shown in bold and underlined.

FIG. 4B shows the nucleotide sequence of the anti-ED-A antibody H1linker sequence (SEQ ID NO: 14).

FIG. 4C shows the nucleotide sequence of the anti-ED-A antibody H1 lightchain (VL) (SEQ ID NO: 13). The nucleotide sequence of the light chainCDR1 of anti-ED-A antibody H1 is underlined. The nucleotide sequence ofthe light chain CDR2 of the anti-ED-A antibody H1 is shown in italicsand underlined. The nucleotide sequence of the light chain CDR3 ofanti-ED-A antibody H1 is shown in bold and underlined.

FIG. 5A shows the amino acid sequence of the anti-ED-A antibody H1 heavychain (VH) (SEQ ID NO: 1). The amino acid sequence of the heavy chainCDR1 (SEQ ID NO: 3) of anti-ED-A antibody H1 is underlined. The aminoacid sequence of the heavy chain CDR2 (SEQ ID NO: 4) of the anti-ED-Aantibody H1 is shown in italics and underlined. The amino acid sequenceof the heavy chain CDR3 (SEQ ID NO: 5) of anti-ED-A antibody H1 is shownin bold and underlined. FIG. 5B shows the amino acid sequence of theanti-ED-A antibody H1 linker sequence (SEQ ID NO: 11).

FIG. 5C shows the amino acid sequence of the anti-ED-A antibody H1 lightchain (VL) (SEQ ID NO: 2). The amino acid sequence of the light chainCDR1 (SEQ ID NO: 6) of anti-ED-A antibody H1 is underlined. The aminoacid sequence of the light chain CDR2 (SEQ ID NO: 7) of the anti-ED-Aantibody H1 is shown in italics and underlined. The amino acid sequenceof the light chain CDR3 (SEQ ID NO: 8) of anti-ED-A antibody H1 is shownin bold and underlined.

FIG. 6 shows the sequence of a nucleic acid construct including a codingsequence for F8-IL10. The structure is HINDIII Secretion sequence F8(14aa linker) linker(SSSSG)₃-IL10-Stop-NotI, as follows: a HINDIIIrestriction site is underlined, sequence encoding the secretion signalis in italics, the F8 VH-encoding sequence is in bold following thesecretion signal sequence, sequence encoding the 14 amino acid linker isin lower case, F8 VL-encoding sequence is in bold following the 14 aminoacid linker sequence, a linker(SSSSG)₃ (amino acids 243-257 of SEQ IDNO: 149) sequence follows the F8 encoding sequence underlined and initalics, the IL-10 encoding sequence is double-underlined; stop is thenin lower case, followed by a NOTI restriction site that is underlined.

FIG. 7 shows the amino acid sequence of an antibody scFv (F8) IL-10conjugate, including linkers, of structure: VH-linker-VL-linker-IL-10.The VH and VL domains are in bold, the scFv linker is in lower case, thelinker between scFv and IL10 is in lower case and italics, the IL-10sequence is underlined.

FIG. 8 illustrates cloning, expression and purification of F8-IL10 andHyHel10-IL10:

FIG. 8 a shows a schematic representation of a pcDNA3.1 vectorcontaining the elements of the F8-IL10 fusion proteins. The human IL10moiety was fused to the C-terminus of the scFv antibody fragment by the15 amino acid linker (SSSSG)3 (amino acids 243-257of SEQ ID NO: 149. Thesecretion sequence at the N-terminus is required for secretion ofrecombinant proteins.

FIG. 8 b shows the results of SDS-PAGE analysis of purified fusionproteins: Lane 1, molecular weight marker; lanes 2 & 3, F8-IL10 undernon-reducing and reducing conditions. The monomeric fusion protein isexpected to have a molecular weight of 46 kDa. FIG. 8 c shows a sizeexclusion chromatography profile of purified F8-IL10 (Superdex 200). Thepeak eluting at 13 ml retention volume corresponds to the non-covalenthomodimeric form of F8-IL10, the smaller peak eluting at 14 ml retentionvolume corresponds to the monomeric fraction.

FIG. 8 d shows the results of an activity assay of F8-IL10. The activityof F8-IL10 was compared with that of recombinant human IL10 on MC/9cells.

TERMINOLOGY

Fibronectin

Fibronectin is an antigen subject to alternative splicing, and a numberof alternative isoforms of fibronectin are known, as described elsewhereherein. Extra Domain-A (EDA or ED-A) is also known as ED, extra type IIIrepeat A (EIIIA) or EDI. The sequence of human ED-A has been publishedby Kornblihtt et al. (1984), Nucleic Acids Res. 12, 5853-5868 andPaolella et al. (1988), Nucleic Acids Res. 16, 3545-3557. The sequenceof human ED-A is also available on the SwissProt database as amino acids1631-1720 (Fibronectin type-III 12; extra domain 2) of the amino acidsequence deposited under accession number P02751. The sequence of mouseED-A is available on the SwissProt database as amino acids 1721-1810(Fibronectin type-III 13; extra domain 2) of the amino acid sequencedeposited under accession number P11276.

The ED-A isoform of fibronectin (A-FN) contains the Extra Domain-A(ED-A). The sequence of the human A-FN can be deduced from thecorresponding human fibronectin precursor sequence which is available onthe SwissProt database under accession number P02751. The sequence ofthe mouse A-FN can be deduced from the corresponding mouse fibronectinprecursor sequence which is available on the SwissProt database underaccession number P11276. The A-FN may be the human ED-A isoform offibronectin. The ED-A may be the Extra Domain-A of human fibronectin.

ED-A is a 90 amino acid sequence which is inserted into fibronectin (FN)by alternative splicing and is located between domain 11 and 12 of FN(Borsi et al., 1987, J. Cell Biol., 104, 595-600). ED-A is mainly absentin the plasma form of FN but is abundant during embryogenesis, tissueremodelling, fibrosis, cardiac transplantation and solid tumour growth.

Alternative Splicing

Alternative splicing refers to the occurrence of different patterns ofsplicing of a primary RNA transcript of DNA to produce different mRNAs.After excision of introns, selection may determine which exons arespliced together to form the mRNA. Alternative splicing leads toproduction of different isoforms containing different exons and/ordifferent numbers of exons. For example one isoform may comprise anadditional amino acid sequence corresponding to one or more exons, whichmay comprise one or more domains.

Binding Member

This describes one member of a pair of molecules that bind one another.The members of a binding pair may be naturally derived or wholly orpartially synthetically produced. One member of the pair of moleculeshas an area on its surface, or a cavity, which binds to and is thereforecomplementary to a particular spatial and polar organization of theother member of the pair of molecules. Examples of types of bindingpairs are antigen-antibody, biotin-avidin, hormone-hormone receptor,receptor-ligand, enzyme-substrate. The present invention is concernedwith antigen-antibody type reactions.

A binding member normally comprises a molecule having an antigen-bindingsite. For example, a binding member may be an antibody molecule or anon-antibody protein that comprises an antigen-binding site.

An antigen binding site may be provided by means of arrangement ofcomplementarity determining regions (CDRs) on non-antibody proteinscaffolds such as fibronectin or cytochrome B etc. (Haan & Maggos, 2004;Koide 1998; Nygren 1997), or by randomising or mutating amino acidresidues of a loop within a protein scaffold to confer bindingspecificity for a desired target. Scaffolds for engineering novelbinding sites in proteins have been reviewed in detail by Nygren et al.(1997). Protein scaffolds for antibody mimics are disclosed inWO/0034784, which is herein incorporated by reference in its entirety,in which the inventors describe proteins (antibody mimics) that includea fibronectin type III domain having at least one randomised loop. Asuitable scaffold into which to graft one or more CDRs, e.g. a set ofHCDRs, may be provided by any domain member of the immunoglobulin genesuperfamily. The scaffold may be a human or non-human protein. Anadvantage of a non-antibody protein scaffold is that it may provide anantigen-binding site in a scaffold molecule that is smaller and/oreasier to manufacture than at least some antibody molecules. Small sizeof a binding member may confer useful physiological properties such asan ability to enter cells, penetrate deep into tissues or reach targetswithin other structures, or to bind within protein cavities of thetarget antigen. Use of antigen binding sites in non-antibody proteinscaffolds is reviewed in Wess, 2004. Typical are proteins having astable backbone and one or more variable loops, in which the amino acidsequence of the loop or loops is specifically or randomly mutated tocreate an antigen-binding site that binds the target antigen. Suchproteins include the IgG-binding domains of protein A from S. aureus,transferrin, tetranectin, fibronectin (e.g. 10th fibronectin type IIIdomain) and lipocalins. Other approaches include synthetic “Microbodies”(Selecore GmbH), which are based on cyclotides—small proteins havingintra-molecular disulphide bonds.

In addition to antibody sequences and/or an antigen-binding site, abinding member for use in the present invention may comprise other aminoacids, e.g. forming a peptide or polypeptide, such as a folded domain,or to impart to the molecule another functional characteristic inaddition to ability to bind antigen. Binding members for use in theinvention may carry a detectable label, or may be conjugated to a toxinor a targeting moiety or enzyme (e.g. via a peptidyl bond or linker).For example, a binding member may comprise a catalytic site (e.g. in anenzyme domain) as well as an antigen binding site, wherein the antigenbinding site binds to the antigen and thus targets the catalytic site tothe antigen. The catalytic site may inhibit biological function of theantigen, e.g. by cleavage.

Although, as noted, CDRs can be carried by non-antibody scaffolds, thestructure for carrying a CDR or a set of CDRs will generally be anantibody heavy or light chain sequence or substantial portion thereof inwhich the CDR or set of CDRs is located at a location corresponding tothe CDR or set of CDRs of naturally occurring VH and VL antibodyvariable domains encoded by rearranged immunoglobulin genes. Thestructures and locations of immunoglobulin variable domains may bedetermined by reference to Kabat 1987, and updates thereof, nowavailable on the Internet (at immuno.bme.nwu.edu or find “Kabat” usingany search engine).

By CDR region or CDR, it is intended to indicate the hypervariableregions of the heavy and light chains of the immunoglobulin as definedby Kabat et al. (1987), (Kabat 1991a, and later editions). An antibodytypically contains 3 heavy chain CDRs and 3 light chain CDRs. The termCDR or CDRs is used here in order to indicate, according to the case,one of these regions or several, or even the whole, of these regionswhich contain the majority of the amino acid residues responsible forthe binding by affinity of the antibody for the antigen or the epitopewhich it recognizes.

Among the six short CDR sequences, the third CDR of the heavy chain(HCDR3) has a greater size variability (greater diversity essentiallydue to the mechanisms of arrangement of the genes which give rise toit). It can be as short as 2 amino acids although the longest size knownis 26. Functionally, HCDR3 plays a role in part in the determination ofthe specificity of the antibody (Segal 1974; Amit 1986; Chothia 1987;Chothia 1989; Caton 1990; Sharon 1990a; Sharon 1990b; Kabat et al.,1991b).

Antibody Molecule

This describes an immunoglobulin whether natural or partly or whollysynthetically produced. The term also relates to any polypeptide orprotein comprising an antibody antigen-binding site. It must beunderstood here that the invention does not relate to the antibodies innatural form, that is to say they are not in their natural environmentbut that they have been able to be isolated or obtained by purificationfrom natural sources, or else obtained by genetic recombination, or bychemical synthesis, and that they can then contain unnatural amino acidsas will be described later. Antibody fragments that comprise an antibodyantigen-binding site include, but are not limited to, antibody moleculessuch as Fab, Fab′, Fab′-SH, scFv, Fv, dAb, Fd; and diabodies.

It is possible to take monoclonal and other antibodies and usetechniques of recombinant DNA technology to produce other antibodies orchimeric molecules that bind the target antigen. Such techniques mayinvolve introducing DNA encoding the immunoglobulin variable region, orthe CDRs, of an antibody to the constant regions, or constant regionsplus framework regions, of a different immunoglobulin. See, forinstance, EP-A-184187, GB 2188638A or EP-A-239400, and a large body ofsubsequent literature. A hybridoma or other cell producing an antibodymay be subject to genetic mutation or other changes, which may or maynot alter the binding specificity of antibodies produced.

As antibodies can be modified in a number of ways, the term “antibodymolecule” should be construed as covering any binding member orsubstance having an antibody antigen-binding site with the requiredspecificity and/or binding to antigen. Thus, this term covers antibodyfragments and derivatives, including any polypeptide comprising anantibody antigen-binding site, whether natural or wholly or partiallysynthetic. Chimeric molecules comprising an antibody antigen-bindingsite, or equivalent, fused to another polypeptide (e.g. derived fromanother species or belonging to another antibody class or subclass) aretherefore included. Cloning and expression of chimeric antibodies aredescribed in EP-A-0120694 and EP-A-0125023, and a large body ofsubsequent literature.

Further techniques available in the art of antibody engineering havemade it possible to isolate human and humanised antibodies. For example,human hybridomas can be made as described by Kontermann & Dubel (2001).Phage display, another established technique for generating bindingmembers has been described in detail in many publications such asWO92/01047 (discussed further below) and U.S. Pat. Nos. 5,969,108,5,565,332, 5,733,743, 5,858,657, 5,871,907, 5,872,215, 5,885,793,5,962,255, 6,140,471, 6,172,197, 6,225,447, 6,291,650, 6,492,160,6,521,404 and Kontermann & Dubel (2001). Transgenic mice in which themouse antibody genes are inactivated and functionally replaced withhuman antibody genes while leaving intact other components of the mouseimmune system, can be used for isolating human antibodies (Mendez 1997).

Synthetic antibody molecules may be created by expression from genesgenerated by means of oligonucleotides synthesized and assembled withinsuitable expression vectors, for example as described by Knappik et al.(2000) or Krebs et al. (2001).

It has been shown that fragments of a whole antibody can perform thefunction of binding antigens. Examples of binding fragments are (i) theFab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fdfragment consisting of the VH and CH1 domains; (iii) the Fv fragmentconsisting of the VL and VH domains of a single antibody; (iv) the dAbfragment (Ward 1989; McCafferty 1990; Holt 2003), which consists of a VHor a VL domain; (v) isolated CDR regions; (vi) F(ab′)₂ fragments, abivalent fragment comprising two linked Fab fragments (vii) single chainFv molecules (scFv), wherein a VH domain and a VL domain are linked by apeptide linker which allows the two domains to associate to form anantigen binding site (Bird 1988; Huston 1988); (viii) bispecific singlechain Fv dimers (PCT/US92/09965) and (ix) “diabodies”, multivalent ormultispecific fragments constructed by gene fusion (WO94/13804; Holliger1993a). Fv, scFv or diabody molecules may be stabilized by theincorporation of disulphide bridges linking the VH and VL domains(Reiter 1996). Minibodies comprising a scFv joined to a CH3 domain mayalso be made (Hu 1996). Other examples of binding fragments are Fab′,which differs from Fab fragments by the addition of a few residues atthe carboxyl terminus of the heavy chain CH1 domain, including one ormore cysteines from the antibody hinge region, and Fab′-SH, which is aFab′ fragment in which the cysteine residue(s) of the constant domainsbear a free thiol group.

Antibody fragments for use in the invention can be obtained startingfrom any of the antibody molecules described herein, e.g. antibodymolecules comprising VH and/or VL domains or CDRs of any of antibodiesdescribed herein, by methods such as digestion by enzymes, such aspepsin or papain and/or by cleavage of the disulfide bridges by chemicalreduction. In another manner, antibody fragments of the presentinvention may be obtained by techniques of genetic recombinationlikewise well known to the person skilled in the art or else by peptidesynthesis by means of, for example, automatic peptide synthesizers suchas those supplied by the company Applied Biosystems, etc., or by nucleicacid synthesis and expression.

Functional antibody fragments according to the present invention includeany functional fragment whose half-life is increased by a chemicalmodification, especially by PEGylation, or by incorporation in aliposome.

A dAb (domain antibody) is a small monomeric antigen-binding fragment ofan antibody, namely the variable region of an antibody heavy or lightchain (Holt 2003). VH dAbs occur naturally in camelids (e.g. camel,llama) and may be produced by immunizing a camelid with a targetantigen, isolating antigen-specific B cells and directly cloning dAbgenes from individual B cells. dAbs are also producible in cell culture.Their small size, good solubility and temperature stability makes themparticularly physiologically useful and suitable for selection andaffinity maturation. A binding member of the present invention may be adAb comprising a VH or VL domain substantially as set out herein, or aVH or VL domain comprising a set of CDRs substantially as set outherein.

As used herein, the phrase “substantially as set out” refers to thecharacteristic(s) of the relevant CDRs of the VH or VL domain of bindingmembers described herein will be either identical or highly similar tothe specified regions of which the sequence is set out herein. Asdescribed herein, the phrase “highly similar” with respect to specifiedregion(s) of one or more variable domains, it is contemplated that from1 to about 5, e.g. from 1 to 4, including 1 to 3, or 1 or 2, or 3 or 4,amino acid substitutions may be made in the CDR and/or VH or VL domain.

Bispecific or bifunctional antibodies form a second generation ofmonoclonal antibodies in which two different variable regions arecombined in the same molecule (Holliger 1999). Their use has beendemonstrated both in the diagnostic field and in the therapy field fromtheir capacity to recruit new effector functions or to target severalmolecules on the surface of tumor cells. Where bispecific antibodies areto be used, these may be conventional bispecific antibodies, which canbe manufactured in a variety of ways (Holliger 1993b), e.g. preparedchemically or from hybrid hybridomas, or may be any of the bispecificantibody fragments mentioned above. These antibodies can be obtained bychemical methods (Glennie 1987; Repp 1995) or somatic methods (Staerz1986; Suresh 1986) but likewise by genetic engineering techniques whichallow the heterodimerization to be forced and thus facilitate theprocess of purification of the antibody sought (Merchand 1998). Examplesof bispecific antibodies include those of the BiTE™ technology in whichthe binding domains of two antibodies with different specificity can beused and directly linked via short flexible peptides. This combines twoantibodies on a short single polypeptide chain. Diabodies and scFv canbe constructed without an Fc region, using only variable domains,potentially reducing the effects of anti-idiotypic reaction.

Bispecific antibodies can be constructed as entire IgG, as bispecificFab′2, as Fab′PEG, as diabodies or else as bispecific scFv. Further, twobispecific antibodies can be linked using routine methods known in theart to form tetravalent antibodies.

Bispecific diabodies, as opposed to bispecific whole antibodies, mayalso be particularly useful because they can be readily constructed andexpressed in E. coli. Diabodies (and many other polypeptides such asantibody fragments) of appropriate binding specificities can be readilyselected using phage display (WO94/13804) from libraries. If one arm ofthe diabody is to be kept constant, for instance, with a specificitydirected against a target antigen, then a library can be made where theother arm is varied and an antibody of appropriate specificity selected.Bispecific whole antibodies may be made by alternative engineeringmethods as described in Ridgeway 1996.

Various methods are available in the art for obtaining antibodiesagainst a target antigen. The antibodies may be monoclonal antibodies,especially of human, murine, chimeric or humanized origin, which can beobtained according to the standard methods well known to the personskilled in the art.

In general, for the preparation of monoclonal antibodies or theirfunctional fragments, especially of murine origin, it is possible torefer to techniques which are described in particular in the manual“Antibodies” (Harlow and Lane 1988) or to the technique of preparationfrom hybridomas described by Kohler and Milstein, 1975.

Monoclonal antibodies can be obtained, for example, from an animal cellimmunized against A-FN, or one of its fragments containing the epitoperecognized by said monoclonal antibodies, e.g. a fragment comprising orconsisting of ED-A, or a peptide fragment of ED-A. The A-FN, or one ofits fragments, can especially be produced according to the usual workingmethods, by genetic recombination starting with a nucleic acid sequencecontained in the cDNA sequence coding for A-FN or fragment thereof, bypeptide synthesis starting from a sequence of amino acids comprised inthe peptide sequence of the A-FN and/or fragment thereof.

Monoclonal antibodies can, for example, be purified on an affinitycolumn on which A-FN or one of its fragments containing the epitoperecognized by said monoclonal antibodies, e.g. a fragment comprising orconsisting of ED-A or a peptide fragment of ED-A, has previously beenimmobilized. Monoclonal antibodies can be purified by chromatography onprotein A and/or G, followed or not followed by ion-exchangechromatography aimed at eliminating the residual protein contaminants aswell as the DNA and the LPS, in itself, followed or not followed byexclusion chromatography on Sepharose gel in order to eliminate thepotential aggregates due to the presence of dimers or of othermultimers. The whole of these techniques may be used simultaneously orsuccessively.

Antigen-binding Site

This describes the part of a molecule that binds to and is complementaryto all or part of the target antigen. In an antibody molecule it isreferred to as the antibody antigen-binding site, and comprises the partof the antibody that binds to and is complementary to all or part of thetarget antigen. Where an antigen is large, an antibody may only bind toa particular part of the antigen, which part is termed an epitope. Anantibody antigen-binding site may be provided by one or more antibodyvariable domains. An antibody antigen-binding site may comprise anantibody light chain variable region (VL) and an antibody heavy chainvariable region (VH).

Isolated

This refers to the state in which binding members for use in theinvention or nucleic acid encoding such binding members, will generallybe in accordance with the present invention. Thus, binding members, VHand/or VL domains of the present invention may be provided isolatedand/or purified, e.g. from their natural environment, in substantiallypure or homogeneous form, or, in the case of nucleic acid, free orsubstantially free of nucleic acid or genes of origin other than thesequence encoding a polypeptide with the required function. Isolatedmembers and isolated nucleic acid will be free or substantially free ofmaterial with which they are naturally associated such as otherpolypeptides or nucleic acids with which they are found in their naturalenvironment, or the environment in which they are prepared (e.g. cellculture) when such preparation is by recombinant DNA technologypractised in vitro or in vivo. Members and nucleic acid may beformulated with diluents or adjuvants and still for practical purposesbe isolated—for example the members will normally be mixed with gelatinor other carriers if used to coat microtitre plates for use inimmunoassays, or will be mixed with pharmaceutically acceptable carriersor diluents when used in diagnosis or therapy. Binding members may beglycosylated, either naturally or by systems of heterologous eukaryoticcells (e.g. CHO or NS0 (ECACC 85110503) cells, or they may be (forexample if produced by expression in a prokaryotic cell) unglycosylated.

Heterogeneous preparations comprising antibody molecules may also beused in the invention. For example, such preparations may be mixtures ofantibodies with full-length heavy chains and heavy chains lacking theC-terminal lysine, with various degrees of glycosylation and/or withderivatized amino acids, such as cyclization of an N-terminal glutamicacid to form a pyroglutamic acid residue.

One or more binding members for an antigen, e.g. the A-FN or the ED-A offibronectin, may be obtained by bringing into contact a library ofbinding members according to the invention and the antigen or a fragmentthereof, e.g. a fragment comprising or consisting of ED-A or a peptidefragment of ED-A and selecting one or more binding members of thelibrary able to bind the antigen.

An antibody library may be screened using Iterative Colony FilterScreening (ICFS). In ICFS, bacteria containing the DNA encoding severalbinding specificities are grown in a liquid medium and, once the stageof exponential growth has been reached, some billions of them aredistributed onto a growth support consisting of a suitably pre-treatedmembrane filter which is incubated until completely confluent bacteriaecolonies appear. A second trap substrate consists of another membranefilter, pre-humidified and covered with the desired antigen.

The trap membrane filter is then placed onto a plate containing asuitable culture medium and covered with the growth filter with thesurface covered with bacterial colonies pointing upwards. The sandwichthus obtained is incubated at room temperature for about 16 h. It isthus possible to obtain the expression of the genes encoding antibodyfragments scFv having a spreading action, so that those fragmentsbinding specifically with the antigen which is present on the trapmembrane are trapped. The trap membrane is then treated to point outbound antibody fragments scFv with colorimetric techniques commonly usedto this purpose.

The position of the coloured spots on the trap filter allows to go backto the corresponding bacterial colonies which are present on the growthmembrane and produced the antibody fragments trapped. Such colonies aregathered and grown and the bacteria—a few millions of them aredistributed onto a new culture membrane repeating the proceduresdescribed above. Analogous cycles are then carried out until thepositive signals on the trap membrane correspond to single positivecolonies, each of which represents a potential source of monoclonalantibody fragments directed against the antigen used in the selection.ICFS is described in e.g. WO0246455, which is incorporated herein byreference. A library may also be displayed on particles or molecularcomplexes, e.g. replicable genetic packages such bacteriophage (e.g. T7)particles, or other in vitro display systems, each particle or molecularcomplex containing nucleic acid encoding the antibody VH variable domaindisplayed on it, and optionally also a displayed VL domain if present.Phage display is described in WO92/01047 and e.g. U.S. Pat. Nos.5,969,108, 5,565,332, 5,733,743, 5,858,657, 5,871,907, 5,872,215,5,885,793, 5,962,255, 6,140,471, 6,172,197, 6,225,447, 6,291,650,6,492,160 and 6,521,404, each of which is herein incorporated byreference in its entirety.

Following selection of binding members able to bind the antigen anddisplayed on bacteriophage or other library particles or molecularcomplexes, nucleic acid may be taken from a bacteriophage or otherparticle or molecular complex displaying a said selected binding member.Such nucleic acid may be used in subsequent production of a bindingmember or an antibody VH or VL variable domain by expression fromnucleic acid with the sequence of nucleic acid taken from abacteriophage or other particle or molecular complex displaying a saidselected binding member.

An antibody VH variable domain with the amino acid sequence of anantibody VH variable domain of a said selected binding member may beprovided in isolated form, as may a binding member comprising such a VHdomain.

Ability to bind the A-FN or the ED-A of fibronectin or other targetantigen or isoform may be further tested, e.g. ability to compete withe.g. any one of anti-ED-A antibodies H1, B2, C5, D5, E5, C8, F8, F1, B7,E8 or G9 for binding to the A-FN or a fragment of the A-FN, e.g. theED-A of fibronectin.

A binding member for use in the invention may bind the A-FN and/or theED-A of fibronectin specifically. A binding member of the presentinvention may bind the A-FN and/or the ED-A of fibronectin with the sameaffinity as anti-ED-A antibody H1, B2, C5, D5, E5, C8, F8, F1, B7, E8 orG9, e.g. in scFv format, or with an affinity that is better. A bindingmember for use in the invention may bind the A-FN and/or the ED-A offibronectin with a K_(D) of 3×10⁻⁸ M or an affinity that is better.Preferably, a binding member for use in the invention binds the A-FNand/or the ED-A of fibronectin with a K_(D) of 2×10⁻⁸ M or an affinitythat is better. More preferably, a binding member for use in theinvention binds the A-FN and/or the ED-A of fibronectin with a K_(D) of1.7×10⁻⁸ M or an affinity that is better. Yet more preferably, a bindingmember for use in the invention binds the A-FN and/or the ED-A offibronectin with a K_(D) of 1.4×10⁻⁸ M or an affinity that is better.Most preferably, a binding member for use in the invention binds theA-FN and/or the ED-A of fibronectin with a K_(D) of 3×10⁻⁹ M or anaffinity that is better.

A binding member of the present invention may bind to the same epitopeon A-FN and/or the ED-A of fibronectin as anti-ED-A antibody H1, B2, C5,D5, E5, C8, F8, F1, B7, E8 or G9.

A binding member for use in the invention may not show any significantbinding to molecules other than the A-FN and/or the ED-A of fibronectin.In particular the binding member may not bind other isoforms offibronectin, for example the ED-β isoform and/or the IIICS isoform offibronectin.

Variants of antibody molecules disclosed herein may be produced and usedin the present invention. The techniques required to make substitutionswithin amino acid sequences of CDRs, antibody VH or VL domains andbinding members generally are available in the art. Variant sequencesmay be made, with substitutions that may or may not be predicted to havea minimal or beneficial effect on activity, and tested for ability tobind A-FN and/or the ED-A of fibronectin and/or for any other desiredproperty.

Variable domain amino acid sequence variants of any of the VH and VLdomains whose sequences are specifically disclosed herein may beemployed in accordance with the present invention, as discussed.Particular variants may include one or more amino acid sequencealterations (addition, deletion, substitution and/or insertion of anamino acid residue), may be less than about 20 alterations, less thanabout 15 alterations, less than about 10 alterations or less than about5 alterations, maybe 5, 4, 3, 2 or 1. Alterations may be made in one ormore framework regions and/or one or more CDRs. The alterations normallydo not result in loss of function, so a binding member comprising athus-altered amino acid sequence may retain an ability to bind A-FNand/or the ED-A of fibronectin. For example, it may retain the samequantitative binding as a binding member in which the alteration is notmade, e.g. as measured in an assay described herein. The binding membercomprising a thus-altered amino acid sequence may have an improvedability to bind A-FN and/or the ED-A of fibronectin.

Novel VH or VL regions carrying CDR-derived sequences for use in theinvention may be generated using random mutagenesis of one or moreselected VH and/or VL genes to generate mutations within the entirevariable domain. In some embodiments one or two amino acid substitutionsare made within an entire variable domain or set of CDRs. Another methodthat may be used is to direct mutagenesis to CDR regions of VH or VLgenes.

As noted above, a CDR amino acid sequence substantially as set outherein may be carried as a CDR in a human antibody variable domain or asubstantial portion thereof. The HCDR3 sequences substantially as setout herein represent embodiments of the present invention and forexample each of these may be carried as a HCDR3 in a human heavy chainvariable domain or a substantial portion thereof.

Variable domains employed in the invention may be obtained or derivedfrom any germ-line or rearranged human variable domain, or may be asynthetic variable domain based on consensus or actual sequences ofknown human variable domains. A variable domain can be derived from anon-human antibody. A CDR sequence for use in the invention (e.g. CDR3)may be introduced into a repertoire of variable domains lacking a CDR(e.g. CDR3), using recombinant DNA technology. For example, Marks et al.(1992) describe methods of producing repertoires of antibody variabledomains in which consensus primers directed at or adjacent to the 5′ endof the variable domain area are used in conjunction with consensusprimers to the third framework region of human VH genes to provide arepertoire of VH variable domains lacking a CDR3. Marks et al. furtherdescribe how this repertoire may be combined with a CDR3 of a particularantibody. Using analogous techniques, the CDR3-derived sequences of thepresent invention may be shuffled with repertoires of VH or VL domainslacking a CDR3, and the shuffled complete VH or VL domains combined witha cognate VL or VH domain to provide binding members for use in theinvention. The repertoire may then be displayed in a suitable hostsystem such as the phage display system of WO92/01047, which is hereinincorporated by reference in its entirety, or any of a subsequent largebody of literature, including Kay, Winter & McCafferty (1996), so thatsuitable binding members may be selected. A repertoire may consist offrom anything from 10⁴ individual members upwards, for example at least10⁹, at least 10⁶, at least 10⁷, at least 10⁸, at least 10⁹ or at least10¹⁰ members.

Similarly, one or more, or all three CDRs may be grafted into arepertoire of VH or VL domains that are then screened for a bindingmember or binding members for the A-FN and/or the ED-A of fibronectin.

One or more of the HCDR1, HCDR2 and HCDR3 of antibody H1, B2, C5, D5,E5, C8, F8, F1, B7, E8 or G9, or the set of HCDRs may be employed,and/or one or more of the X LCDR1, LCDR2 and LCDR3 of antibody H1, B2,C5, D5, E5, C8, F8, F1, B7, E8 or G9 or the set of LCDRs of antibody H1,B2, C5, D5, E5, C8, F8, F1, B7, E8 or G9 may be employed.

Similarly, other VH and VL domains, sets of CDRs and sets of HCDRsand/or sets of LCDRs disclosed herein may be employed.

The A-FN and/or the ED-A of fibronectin may be used in a screen forbinding members, e.g. antibody molecules, for use in the preparation ofa medicament for the treatment of rheumatoid arthritis. The screen may ascreen of a repertoire as disclosed elsewhere herein.

A substantial portion of an immunoglobulin variable domain may compriseat least the three CDR regions, together with their interveningframework regions. The portion may also include at least about 50% ofeither or both of the first and fourth framework regions, the 50% beingthe C-terminal 50% of the first framework region and the N-terminal 50%of the fourth framework region. Additional residues at the N-terminal orC-terminal end of the substantial part of the variable domain may bethose not normally associated with naturally occurring variable domainregions. For example, construction of binding members of the presentinvention made by recombinant DNA techniques may result in theintroduction of N- or C-terminal residues encoded by linkers introducedto facilitate cloning or other manipulation steps. Other manipulationsteps include the introduction of linkers to join variable domainsdisclosed elsewhere herein to further protein sequences includingantibody constant regions, other variable domains (for example in theproduction of diabodies) or detectable/functional labels as discussed inmore detail elsewhere herein.

Although binding members may comprise a pair of VH and VL domains,single binding domains based on either VH or VL domain sequences mayalso be used in the invention. It is known that single immunoglobulindomains, especially VH domains, are capable of binding target antigensin a specific manner. For example, see the discussion of dAbs above.

In the case of either of the single binding domains, these domains maybe used to screen for complementary domains capable of forming atwo-domain binding member able to bind A-FN and/or the ED-A offibronectin. This may be achieved by phage display screening methodsusing the so-called hierarchical dual combinatorial approach asdisclosed in WO92/01047, herein incorporated by reference in itsentirety, in which an individual colony containing either an H or Lchain clone is used to infect a complete library of clones encoding theother chain (L or H) and the resulting two-chain binding member isselected in accordance with phage display techniques such as thosedescribed in that reference. This technique is also disclosed in Marks1992.

Binding members for use in the present invention may further compriseantibody constant regions or parts thereof, e.g. human antibody constantregions or parts thereof. For example, a VL domain may be attached atits C-terminal end to antibody light chain constant domains includinghuman Cκ or Cλ chains, e.g. Cλ. Similarly, a binding member based on aVH domain may be attached at its C-terminal end to all or part (e.g. aCH1 domain) of an immunoglobulin heavy chain derived from any antibodyisotype, e.g. IgG, IgA, IgE and IgM and any of the isotype sub-classes,particularly IgG1 and IgG4. Any synthetic or other constant regionvariant that has these properties and stabilizes variable regions isalso useful in embodiments of the present invention.

Binding members for use in the invention may be labelled with adetectable or functional label. A label can be any molecule thatproduces or can be induced to produce a signal, including but notlimited to fluorescers, radiolabels, enzymes, chemiluminescers orphotosensitizers. Thus, binding may be detected and/or measured bydetecting fluorescence or luminescence, radioactivity, enzyme activityor light absorbance. Detectable labels may be attached to antibodies foruse in the invention using conventional chemistry known in the art.

There are numerous methods by which the label can produce a signaldetectable by external means, for example, by visual examination,electromagnetic radiation, heat, and chemical reagents. The label canalso be bound to another binding member that binds the antibody for usein the invention, or to a support.

Labelled binding members, e.g. scFv labelled with a detectable label,may be used diagnostically in vivo, ex vivo or in vitro, and/ortherapeutically.

For example, radiolabelled binding members (e.g. binding membersconjugated to a radioisotope) may be used in radiodiagnosis andradiotherapy. Radioisotopes which may be conjugated to a binding memberfor use in the invention include isotopes such as ^(94m)Tc, ^(99m)Tc,¹⁸⁶Re, ¹⁸⁸Re, ²⁰³Pb, ⁶⁷Ga, ⁶⁸Ga, ⁴⁷Sc, ¹¹¹In, ⁹⁷Ru, ⁶²Cu, ⁶⁴Cu, ⁸⁶Y,⁸⁸Y, ⁹⁰Y, ¹²¹Sn, ¹⁶¹Tb, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁰⁵Rh, ¹⁷⁷Lu, ¹²³I, ¹²⁴I, ¹²⁵I and¹³¹I.

For example, a binding member for use in the invention labelled with adetectable label may be used to detect, diagnose or monitor rheumatoidarthritis in a human or animal.

A binding member of the present invention may be used for themanufacture of a diagnostic product for use in diagnosing rheumatoidarthritis.

The present invention provides a method of detecting or diagnosingrheumatoid arthritis in a human or animal comprising:

-   -   (a) administering to the human or animal a binding member of the        present invention, for example labelled with a detectable label,        which binds the ED-A isoform of fibronectin and/or the ED-A of        fibronectin, and    -   (b) determining the presence or absence of the binding member in        neovasculature of the human or animal body;        wherein localisation of the binding member to neovasculature in        the human or animal is indicative of the presence of rheumatoid        arthritis.

Where the binding member is labelled with a detectable label, thepresence or absence of the detectable label may be determined bydetecting the label.

A conjugate or fusion between a binding member for use in the inventionand a molecule that exerts a biocidal, cytotoxic immunosuppressive oranti-inflammatory effect on target cells in the lesions and an antibodydirected against an extracellular matrix component which is present insuch lesions may be employed in the present invention. For example, theconjugated molecule may be inter alia interleukin-10, ananti-inflammatory or other drug, a photosensitizer or a radionuclide.Such conjugates may be used therapeutically, e.g. for treatment ofrheumatoid arthritis as referred to herein.

Production and use of fusions or conjugates of binding members withbiocidal or cytotoxic molecules is described for example in WO01/62298,which is incorporated by reference herein.

The invention provides a method of treating rheumatoid arthritis, themethod comprising administering to an individual a therapeuticallyeffective amount of a medicament comprising a binding member for use inthe invention.

The binding member may be a conjugate of (i) a molecule which exerts ananti-inflammatory effect on target cells by cellular interaction, ananti-inflammatory molecule, IL-10, TGF beta, or other drug, and (ii) abinding member for the ED-A isoform of fibronectin and/or the ED-A offibronectin.

The binding member may be a conjugate of (i) a molecule which exerts animmunosuppressive or anti-inflammatory effect and (ii) a binding memberfor the ED-A isoform of fibronectin and/or the ED-A of fibronectin.

The binding member may be a conjugate of (i) interleukin-10 (IL10) orTGF beta and (ii) a binding member for the ED-A isoform of fibronectinand/or the ED-A of fibronectin. Such a binding member is useful inaspects of the invention disclosed herein relating to treatment ofrheumatoid arthritis.

The invention provides the use of a binding member for use in theinvention for the preparation of a medicament for the treatment ofrheumatoid arthritis.

The binding member may be a conjugated or fused to a molecule thatexerts a biocidal, cytotoxic, immunosuppressive or anti-inflammatoryeffect as described herein. The binding member may be a conjugate of (i)a molecule which exerts a biocidal or cytotoxic effect on target cellsby cellular interaction or has an immunosuppressive or anti-inflammatoryeffect and (ii) a binding member for human fibronectin according to thepresent invention.

Also described herein is a conjugate of (i) a molecule which exerts abiocidal or cytotoxic effect on target cells by cellular interaction, oran immunosuppressive or anti-inflammatory effect and (ii) a bindingmember for human fibronectin according for use in the present invention.Such a conjugate preferably comprises a fusion protein comprising thebiocidal, cytotoxic, immunosuppressive or anti-inflammatory molecule anda said binding member, or, where the binding member is two-chain ormulti-chain, a fusion protein comprising the biocidal, cytotoxic,immunosuppressive or anti-inflammatory molecule and a polypeptide chaincomponent of said binding member. Preferably the binding member is asingle-chain polypeptide, e.g. a single-chain antibody molecule, such asscFv.

A fusion protein comprising the immunosuppressive or anti-inflammatorymolecule and a single-chain Fv antibody molecule may be used in theinvention.

The immunosuppressive or anti-inflammatory molecule that exerts itseffect on target cells by cellular interaction, may interact directlywith the target cells, may interact with a membrane-bound receptor onthe target cell or perturb the electrochemical potential of the cellmembrane. In an exemplary preferred embodiment the molecule is IL-10.

As discussed further below, the specific binding member is preferably anantibody or comprises an antibody antigen-binding site. Conveniently,the specific binding member may be a single-chain polypeptide, such as asingle-chain antibody. This allows for convenient production of a fusionprotein comprising single-chain antibody and immunosuppressive oranti-inflammatory molecule (e.g. interleukin-10 or TGF beta. An antibodyantigen-binding site may be provided by means of association of anantibody VH domain and an antibody VL domain in separate polypeptides,e.g. in a complete antibody or in an antibody fragment such as Fab ordiabody. Where the specific binding member is a two-chain or multi-chainmolecule (e.g. Fab or whole antibody, respectively), theimmunosuppressive or anti-inflammatory molecule may be conjugated as afusion polypeptide with one or more polypeptide chains in the specificbinding member.

The binding member may be conjugated with the immunosuppressive oranti-inflammatory molecule by means of a peptide bond, i.e. within afusion polypeptide comprising said molecule and the specific bindingmember or a polypeptide chain component thereof (see e.g. Trachsel etal.). Other means for conjugation include chemical conjugation,especially cross-linking using a bifunctional reagent (e.g. employingDOUBLE-REAGENTS™ Cross-linking Reagents Selection Guide, Pierce).

Also described herein is isolated nucleic acid encoding a binding memberfor use in the present invention. Nucleic acid may include DNA and/orRNA. A nucleic acid may code for a CDR or set of CDRs or VH domain or VLdomain or antibody antigen-binding site or antibody molecule, e.g. scFvor IgG, e.g. IgG1, as defined above. The nucleotide sequences may encodethe VH and/or VL domains disclosed herein.

Further described herein are constructs in the form of plasmids,vectors, transcription or expression cassettes which comprise at leastone polynucleotide as described above.

A recombinant host cell that comprises one or more constructs as aboveare also described. A nucleic acid encoding any CDR or set of CDRs or VHdomain or VL domain or antibody antigen-binding site or antibodymolecule, e.g. scFv or IgG1 or IgG4 as provided, is described, as is amethod of production of the encoded product, which method comprisesexpression from encoding nucleic acid. Expression may conveniently beachieved by culturing under appropriate conditions recombinant hostcells containing the nucleic acid. Following production by expression aVH or VL domain, or binding member may be isolated and/or purified usingany suitable technique, then used as appropriate.

A nucleic acid may comprise DNA or RNA and may be wholly or partiallysynthetic. Reference to a nucleotide sequence as set out hereinencompasses a DNA molecule with the specified sequence, and encompassesa RNA molecule with the specified sequence in which U is substituted forT, unless context requires otherwise.

A method of production of an antibody VH variable domain, the methodincluding causing expression from encoding nucleic acid is alsodescribed. Such a method may comprise culturing host cells underconditions for production of said antibody VH variable domain.

A method of production may comprise a step of isolation and/orpurification of the product. A method of production may compriseformulating the product into a composition including at least oneadditional component, such as a pharmaceutically acceptable excipient.

Systems for cloning and expression of a polypeptide in a variety ofdifferent host cells are well known. Suitable host cells includebacteria, mammalian cells, plant cells, filamentous fungi, yeast andbaculovirus systems and transgenic plants and animals. The expression ofantibodies and antibody fragments in prokaryotic cells is wellestablished in the art. For a review, see for example Plückthun 1991. Acommon bacterial host is E. coli.

Expression in eukaryotic cells in culture is also available to thoseskilled in the art as an option for production of a binding member forexample Chadd & Chamow (2001), Andersen & Krummen (2002), Larrick &Thomas (2001). Mammalian cell lines available in the art for expressionof a heterologous polypeptide include Chinese hamster ovary (CHO) cells,HeLa cells, baby hamster kidney cells, NS0 mouse melanoma cells, YB2/0rat myeloma cells, human embryonic kidney cells, human embryonic retinacells and many others.

Suitable vectors can be chosen or constructed, containing appropriateregulatory sequences, including promoter sequences, terminatorsequences, polyadenylation sequences, enhancer sequences, marker genesand other sequences as appropriate. Vectors may be plasmids e.g.phagemid, or viral e.g. ‘phage’ as appropriate. For further details see,for example, Sambrook & Russell (2001). Many known techniques andprotocols for manipulation of nucleic acid, for example in preparationof nucleic acid constructs, mutagenesis, sequencing, introduction of DNAinto cells and gene expression, and analysis of proteins, are describedin detail in Ausubel 1999.

A host cell may contain a nucleic acid as described herein. Such a hostcell may be in vitro and may be in culture. Such a host cell may be invivo. In vivo presence of the host cell may allow intracellularexpression of a binding member for use in the present invention as“intrabodies” or intracellular antibodies. Intrabodies may be used forgene therapy.

A method comprising introducing a nucleic acid disclosed herein into ahost cell is also described. The introduction may employ any availabletechnique. For eukaryotic cells, suitable techniques may include calciumphosphate transfection, DEAE-Dextran, electroporation, liposome-mediatedtransfection and transduction using retrovirus or other virus, e.g.vaccinia or, for insect cells, baculovirus. Introducing nucleic acid inthe host cell, in particular a eukaryotic cell may use a viral or aplasmid based system. The plasmid system may be maintained episomally ormay be incorporated into the host cell or into an artificial chromosome.Incorporation may be either by random or targeted integration of one ormore copies at single or multiple loci. For bacterial cells, suitabletechniques may include calcium chloride transformation, electroporationand transfection using bacteriophage.

The introduction may be followed by causing or allowing expression fromthe nucleic acid, e.g. by culturing host cells under conditions forexpression of the gene. The purification of the expressed product may beachieved by methods known to one of skill in the art.

The nucleic acid may be integrated into the genome (e.g. chromosome) ofthe host cell. Integration may be promoted by inclusion of sequencesthat promote recombination with the genome, in accordance with standardtechniques.

A method that comprises using a construct as stated above in anexpression system in order to express a binding member or polypeptide asabove is also described.

Binding members for use in the present invention are designed to be usedin methods of diagnosis or treatment in human or animal subjects, e.g.human. Binding members for use in the invention may be used in diagnosisor treatment of rheumatoid arthritis.

Accordingly, the invention provides methods of treatment comprisingadministration of a binding member as provided, pharmaceuticalcompositions comprising such a binding member, and use of such a bindingmember in the manufacture of a medicament for administration, forexample in a method of making a medicament or pharmaceutical compositioncomprising formulating the binding member with a pharmaceuticallyacceptable excipient. Pharmaceutically acceptable vehicles are wellknown and will be adapted by the person skilled in the art as a functionof the nature and of the mode of administration of the activecompound(s) chosen.

Binding members for use in the present invention will usually beadministered in the form of a pharmaceutical composition, which maycomprise at least one component in addition to the binding member. Thuspharmaceutical compositions according to the present invention, and foruse in accordance with the present invention, may comprise, in additionto active ingredient, a pharmaceutically acceptable excipient, carrier,buffer, stabilizer or other materials well known to those skilled in theart. Such materials should be non-toxic and should not interfere withthe efficacy of the active ingredient. The precise nature of the carrieror other material will depend on the route of administration, which maybe oral, inhaled or by injection, e.g. intravenous.

Pharmaceutical compositions for oral administration such as for examplenanobodies etc are also envisaged in the present invention. Such oralformulations may be in tablet, capsule, powder, liquid or semi-solidform. A tablet may comprise a solid carrier such as gelatin or anadjuvant. Liquid pharmaceutical compositions generally comprise a liquidcarrier such as water, petroleum, animal or vegetable oils, mineral oilor synthetic oil. Physiological saline solution, dextrose or othersaccharide solution or glycols such as ethylene glycol, propylene glycolor polyethylene glycol may be included.

For intravenous injection, or injection at the site of affliction, theactive ingredient will be in the form of a parenterally acceptableaqueous solution which is pyrogen-free and has suitable pH, isotonicityand stability. Those of relevant skill in the art are well able toprepare suitable solutions using, for example, isotonic vehicles such asSodium Chloride Injection, Ringer's Injection, Lactated Ringer'sInjection. Preservatives, stabilizers, buffers, antioxidants and/orother additives may be employed, as required. Many methods for thepreparation of pharmaceutical formulations are known to those skilled inthe art. See e.g. Robinson, 1978.

A composition may be administered alone or in combination with othertreatments, concurrently or sequentially or as a combined preparationwith another therapeutic agent or agents, dependent upon the conditionto be treated.

A binding member for use in the present invention may be used as part ofa combination therapy in conjunction with an additional medicinalcomponent. Combination treatments may be used to provide significantsynergistic effects, particularly the combination of a binding memberfor use in the present invention with one or more other drugs. A bindingmember for use in the present invention may be administered concurrentlyor sequentially or as a combined preparation with another therapeuticagent or agents, for the treatment of one or more of the conditionslisted herein.

For example, a binding member for use in the invention may be used incombination with an existing therapeutic agent for the treatment ofrheumatoid arthritis.

Existing therapeutic agents for the treatment of rheumatoid arthritisinclude IL-10, TGFbeta, photosensitizers and cytotoxic drugs.

A binding member for use in the invention and one or more of the aboveadditional medicinal components may be used in the manufacture of amedicament. The medicament may be for separate or combinedadministration to an individual, and accordingly may comprise thebinding member and the additional component as a combined preparation oras separate preparations. Separate preparations may be used tofacilitate separate and sequential or simultaneous administration, andallow administration of the components by different routes e.g. oral andparenteral administration.

In accordance with the present invention, compositions provided may beadministered to mammals. Administration may be in a “therapeuticallyeffective amount”, this being sufficient to show benefit to a patient.Such benefit may be at least amelioration of at least one symptom. Thus“treatment of rheumatoid arthritis” refers to amelioration of at leastone symptom. The actual amount administered, and rate and time-course ofadministration, will depend on the nature and severity of what is beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the composition, the type of binding member, the method ofadministration, the scheduling of administration and other factors knownto medical practitioners. Prescription of treatment, e.g. decisions ondosage etc, is within the responsibility of general practitioners andother medical doctors, and may depend on the severity of the symptomsand/or progression of a disease being treated. Appropriate doses ofantibody are well known in the art (Ledermann 1991 and Bagshawe 1991.Specific dosages indicated herein, or in the Physician's Desk Reference(2003) as appropriate for the type of medicament being administered, maybe used. A therapeutically effective amount or suitable dose of abinding member for use in the invention can be determined by comparingits in vitro activity and in vivo activity in an animal model. Methodsfor extrapolation of effective dosages in mice and other test animals tohumans are known. The precise dose will depend upon a number of factors,including whether the antibody is for diagnosis, prevention or fortreatment, the size and location of the area to be treated, the precisenature of the antibody (e.g. whole antibody, fragment or diabody), andthe nature of any detectable label or other molecule attached to theantibody. A typical antibody dose will be in the range 100 μg to 1 g forsystemic applications, and 1 μg to 1 mg for topical applications. Aninitial higher loading dose, followed by one or more lower doses, may beadministered. An antibody may be a whole antibody, e.g. the IgG1 or IgG4isotype. This is a dose for a single treatment of an adult patient,which may be proportionally adjusted for children and infants, and alsoadjusted for other antibody formats in proportion to molecular weight.Treatments may be repeated at daily, twice-weekly, weekly or monthlyintervals, at the discretion of the physician. Treatments may be everytwo to four weeks for subcutaneous administration and every four toeight weeks for intravenous administration. In some embodiments of thepresent invention, treatment is periodic, and the period betweenadministrations is about two weeks or more, e.g. about three weeks ormore, about four weeks or more, or about once a month. In otherembodiments of the invention, treatment may be given before, and/orafter surgery, and may be administered or applied directly at theanatomical site of surgical treatment.

Further aspects and embodiments of the invention will be apparent tothose skilled in the art given the present disclosure including thefollowing experimental exemplification.

EXPERIMENTAL

Results

Histochemical Analysis of Human Arthritic Specimens

Expression of fibronectin domains EDA and EDB as well as tenascin-Cdomains A1 and C were investigated by immunohistochemistry on humanarthritic specimens using the F8, L19, F16 and G11 antibodiesrespectively.

In FIG. 1 darker staining indicates expression of the respectiveantigens (indicated with white arrows). The anti-EDA antibody F8 led tothe strongest staining, therefore all further experiments were performedwith this antibody.

Immunofluorescence experiments with the F8 antibody were performed whichshowed a nice perivascular staining (visible as white structures in FIG.2).

The human monoclonal antibody F8 selectively accumulates at sites ofarthritis in mice

We studied the in vivo targeting performance of F8 in mini-antibodyformat (SIP) (Borsi et al. 2002) in the CIA mouse model (Courtney et al.1980) using both fluorescence and radioactivity for antibody detection.The SIP format consists of a scFv antibody fragment linked to the CH4domain of human IgE giving rise to a homodimeric protein of 80 kDa insize.

Arthritic mice were injected with SIP(F8) labelled with thenear-infrared dye Alexa 750. Twenty-four hours after intravenousinjection, animals were imaged using an infrared fluorescence imager(Birchler et al., 1999), revealing a strong and selective antibodyaccumulation in the lesions present in the arthritic limb, visible aswhite lighting paws, with some grade 2 swelling in front paws of themice.

Twenty-four hours after intravenous injection of SIP(F8) radioactivelylabelled with ¹²⁵I, mice were sacrificed and paws imaged byautoradiography (phosphorimaging). A preferential accumulation ofradioactivity was observed in the inflamed extremities of mice injectedwith SIP(F8), visible as black staining in autoradiography. One pawshowed an arthritic score of 2 (swelling of the whole paw). Another pawwas classified as grade 1 arthritis (swelling of single fingers).

Activity of Anti-Ed-A Antibody-Interleukin-10 Fusion

Antibody molecule F8 in scFv format was conjugated within a fusionprotein with interleukin-10 (IL-10). Biological activity of the fusionprotein was compared with that of human IL-10 in an assay determiningability to induce IL-4 dependent proliferation of MC/9 cells (Thompsonet al., 1991). The results are shown in FIG. 8( d).

Materials and Methods

Immunohistochemical Analysis on Human Arthritic Specimens

Frozen sections of human arthritic specimens were fixed in ice-coldacetone for 10′, blocked with Fetal Bovine Serum for 30′ and stained formarkers of neovasculature (Fibronectin ED-A and ED-B, Tenascin-C domainA1 and C). The F8, L19, F16 and G11 antibodies were used as myc-taggedscFvs in a concentration of 10 ug/ml and incubated for 1 h. The primaryantibodies were coincubated with the anti-myc antibody 9E10 inconcentration of 7 ug/ml. As tertiary detection antibody a rabbitanti-mouse IgG antibody (Dako, Denmark) and APAAP Mouse Monoclonal(Dako, Denmark) were used in concentrations of 5 and 50 ug/mlrespectively for 1 h each. Fast Red Tablets (Sigma, Switzerland) wereused to develop the staining incubating for 15′. Slides werecounterstained with hematoxylin for 2′, washed with water, mounted withGlycergel mounting medium (Dako, Denmark) and analyzed with a AxiovertS100 TV microscope (Zeiss, Switzerland).

Immunofluorescence Analysis on Human Arthritic Specimens

Frozen sections of human arthritic specimens were fixed in ice-coldacetone for 10′, blocked with Fetal Bovine Serum for 30′ and stained forthe EDA domain of fibronectin. The F8 antibody was used as a myc-taggedscFv in a concentration of 10 ug/ml and incubated for 1 h. The primaryantibody was coincubated with the anti-myc antibody 9E10 in aconcentration of 7 ug/ml. As tertiary detection antibody a fluorescentanti-mouse-Alexa 596 antibody (Molecular Probes, Denmark) was used in aconcentration of 10 ug/ml for 1 h each. Slides were counterstained withHoechst 33342, mounted with Glycergel mounting medium (Dako, Denmark)and analyzed with a AxioScop 2MOT+ microscope (Zeiss, Switzerland).

Animal Model

Male DBA/1 mice (8-12 weeks old) were immunized by intradermal injectionat the base of the tail of 200 μg of bovine type II collagen (MDBiosciences) emulsified with equal volumes of Freund's complete adjuvant(MD Biosciences). 2 weeks after the first immunization the procedure wasrepeated but incomplete Freund's adjuvant (MD Biosciences) was used toemulsify the collagen. Mice were inspected daily and each mouse thatexhibited erythema and/or paw swelling in 1 or more limbs was assignedfor imaging or treatment studies.

Arthritis was monitored using 2 disease indices (clinical score and pawswelling). For the clinical score each limb was graded daily in a notblinded fashion. (0=normal, 1=swelling of 1 or more fingers of the samelimb, 2=swelling of the whole paw), resulting in a maximum possiblescore of 8 per animal. Paw swelling was assessed every second day usinga calliper to measure the thickness of each limb under isofluraneanaesthesia. The mean value of all 4 paws was assigned as paw thicknessto each animal.

Near-Infrared-Imaging of Arthritic Paws

The selective accumulation of SIP(F8) in arthritic mice was tested byNear-Infrared-Imaging analysis as described by Birchler et al. (1999).Briefly, purified SIP(F8) was labelled with Alexa750 (MolecularProbes,Leiden, The Netherlands) according to the manufacturer's recommendationsand 100 ug of labelled protein were injected into the tail vein ofarthritic mice. Mice were anesthetized with Ketamin 80 mg/kg andMedetomidin 0.2 mg/kg and imaged in a near-infrared-mouseimager 24 hrafter injection (Trachsel et al. 2007; Birchler et al. 1999).

Biodistribution Experiments

The in vivo targeting performance of SIP(F8) in arthritic mice wasevaluated by biodistribution analysis as described before (Borsi et al.2002; Tarli et al., 1999). Briefly, purified SIP(F8) was radioiodinatedand 10 ug of protein, corresponding to 11uCi ¹²⁵I, were injected intothe tail vein of arthritic mice. Mice were sacrificed 24 hr afterinjection and paws were exposed for 1 hour and read in a phosphorimager(Fujifilm BAS-5000) as described before (Trachsel et al. 2007).

Antibodies

The isolation of the anti-ED-B antibody fragment scFv(L19) has beenpreviously described (Pini et al. 1998). The parent anti-ED-A antibodywas isolated from the ETH-2 library using published procedures(Giovannoni, Nucleic. Acid Research, 2001, 29(5):E27). The affinitymaturation of the parent anti-ED-A antibody, yielding the high affinityanti-ED-A antibodies, is described in the following section.

Affinity Maturation of the Parent Anti-ED-A Antibody

The parent anti-ED-A antibody (an ETH-2-derived antibody) was used astemplate for the construction of an affinity maturation library.Sequence variability in the VH CDR1 (DP47 germline) and VL CDR1 (DPK22germline) of the library was introduced by PCR using partiallydegenerate primers5′-CTGGAGCCTGGCGGACCCAGCTCATMNNMNNMNNGCTAAAGGTGAATCCAGA-3′ (SEQ ID NO:17) for VH and5′-CCAGGTTTCTGCTGGTACCAGGCTAAMNNMNNMNNGCTAACACTCTGACTGGCCCTGC-3′ (SEQ IDNO: 18) for VL (all oligonucleotides were purchased from OperonBiotechnologies, Cologne, Germany), in a process that generates randommutations at positions 31, 32 and 33 of the VH CDR1 and at positions 31,31a and 32 of the VL CDR1. VHVL combinations were assembled in scFvformat by PCR assembly using the primers LMB3long(5′-CAGGAAACAGCTATGACCATGATTAC-3′) (SEQ ID NO: 19) and fdseqlong(5′-GACGTTAGTAAATGAATTTTCTGTATGAGG-3′) (SEQ ID NO: 20), usinggel-purified VH and VL segments as templates. The assembled VH-VLfragments were doubly digested with NcoI/NotI and cloned intoNcoI/NotI-digested pHEN1 phagemid vector (Hoogenboom et al., 1991). Theresulting ligation product was electroporated into electrocompetent E.coli TG-1 cells according to (Viti et al., 2000), giving rise to alibrary containing 1.5×10⁷ individual antibody clones, which wasscreened for antibodies which bind ED-A with improved affinity.

Selection of Anti-ED-A Antibodies

The antibody library described above was screened for antibodies whichbound ED-A with a greater affinity than the parent anti-ED-A antibodyusing BIAcore analysis. The antigen (11A12) used in the BIAcore analysiscontained the ED-A domain of human fibronectin and has the followingamino acid sequence (SEQ ID NO: 120):

MRSYRTEIDKPSQMQVTDVQDNSISVKWLPSSSPVTGYRVTTTPKNGPGPTKTKTAGPDQTEMTIEGLQPTVEYVVSVYAQNPSGESQPLVQTAVTNIDRPKGLAFTDVDVDSIKIAWESPQGQVSRYRVTYSSPEDGIHELFPAPDGEEDTAELQGLRPGSEYTVSVVALHDDMESQPLIGTQSTAIPAPTDLKFTQVTPTSLSAQWTPPNVQLTGYRVRVTPKEKTGPMKEINLAPDSSSVVVSGLMVATKYEVSVYALKDTLTSRPAQGVVTTLENVRSHHHHHH

The nucleotide sequence of antigen (11A12) (SEQ ID NO: 121) is asfollows:

atgagatcctaccgaacagaaattgacaaaccatcccagatgcaagtgaccgatgttcaggacaacagcattagtgtcaagtggctgccttcaagttcccctgttactggttacagagtaaccaccactcccaaaaatggaccaggaccaacaaaaactaaaactgcaggtccagatcaaacagaaatgactattgaaggcttgcagcccacagtggagtatgtggttagtgtctatgctcagaatccaagcggagagagtcagcctctggttcagactgcagtaaccaacattgatcgccctaaaggactggcattcactgatgtggatgtcgattccatcaaaattgcttgggaaagcccacaggggcaagtttccaggtacagggtgacctactcgagccctgaggatggaatccatgagctattccctgcacctgatggtgaagaagacactgcagagctgcaaggcctcagaccgggttctgagtacacagtcagtgtggttgccttgcacgatgatatggagagccagcccctgattggaacccagtccacagctattcctgcaccaactgacctgaagttcactcaggtcacacccacaagcctgagcgcccagtggacaccacccaatgttcagctcactggatatcgagtgcgggtgacccccaaggagaagaccggaccaatgaaagaaatcaaccttgctcctgacagctcatccgtggttgtatcaggacttatggtggccaccaaatatgaagtgagtgtctatgctcttaaggacactttgacaagcagaccagctcagggagttgtcaccactctggagaatgtcagatctcatc accatcaccatcactaa

The nucleotide sequence of the antigen was amplified by PCR usingprimers containing BamHI and BglII restriction sites at the 5′ and 3′respectively. The resulting PCR product and the vector pQE12 (QIAGEN)were digested with BamHI and BglII restriction endonuclease andsubsequently ligated in a reaction containing a ratio of insert tovector of 3:1. The resulting vector was sequenced to check that thesequence was correct.

Antigen Preparation

A TG1 electrocompetent Preculture in 10 ml 2TY, Amp, 1% Glucose waselectroporated in the presence of 1 μl of a DNA miniprep of 11A12. Thepre-culture was then diluted 1:100 (8 ml in 800 ml of 2TY, Amp, 0.1%Glucose) and grown to an OD600 of 0.4-0.6 and then induced with IPTGover night. The following day the cells were spun down and thesupernatant filtered (Millipore 0.22 μm). After centrifugation andclarification of the culture broth, 11A12 was purified using a Hitrapcolumn on FPLC. The Ni/column was regenerated as follows: the column wasrinsed with 5 column volumes (CV) H2O followed by application of 3CV 0.5M EDTA/0.2 M Tris pH 8 to wash the old Nickel out from the column. Thiswas followed by rinsing of the column with 5CV H2O. The column was thenreloaded with 2CV 100 mM NiSO4 followed by rinsing of the column withseveral CVs H2O. The column was then equilibrated with 5CV lysis buffer(20 mM imidazol/250 mM NaCl/PBS pH 7.4). The cell lysate was filtered(Millipore 0.45 μm) and loaded onto the column (manually). The columnwas then put back on FPLC and the lysis buffer left to flow until the UVsignal was stable (constant), about 3 CV. The elution program was thenstarted: Gradient from 0% to 100% of Elution Buffer (400 mM imidazol/250mM NaCl/PBS pH 7.4) in 5CV. The fractions containing the eluted antigenwere pooled and dialysed in PBS over night.

Expression and Purification of the Anti-ED-A Antibodies

The anti-ED-A antibodies were expressed and purified as follows: A TG1electrocompetent Preculture in 10 ml 2TY, Amp, 1% Glucose waselectroporated in the presence of 1 μl of a DNA miniprep of one of theanti-ED-A antibodies. The pre-culture was then diluted 1:100 (8 ml in800 ml of 2TY, Amp, 0.1% Glucose) and grown to an OD600 of 0.4-0.6 andthen induced with IPTG over night. The following day the cells were spundown and the supernatant filtered (Millipore 0.22 μm). The scFv werepurified on a Protein A-Sepharose column and Triethylemmine was used toelute the scFvs from the column. The fractions containing the elutedscFvs were dialysed in PBS over night at 4° C. The scFv fractions werethen put on a Superdex 75 column with PBS flowing at 0.5 ml/min and 0.25ml fractions collected. The monomeric fractions were used for BIAcoreanalysis.

BIAcore™ Analysis 1

The BIAcore™ Chip was flushed overnight at a flow rate of 5 μl/min withHBS-EP buffer BIACORE™, 0.01 M Hepes pH 7.4, 0.15 M NaCl, 3 mM EDTA,0.005% surfactant P20 (same buffer used for the assay). The antigen(11A12) was diluted to a concentration of 50 μg/ml in acetate buffer (pH4.0) and the COOH groups on the chip were activated by injection of 50μl of a mix of N-Hydroxy Succinimmide (NHS) andethyl-N-(dimethylaminopropyl)-carbodiimide (EDC). 40 μl of the 11A12antigen were injected onto the chip and the residual free COOH groupswere blocked with 30 μl of ethanolamine. After a 0.22 μm filtration, 20μl of each individual bacterial supernatant were injected onto the chipand interaction with the antigen was monitored in real time.

BIAcore™ Analysis 2

The k_(on), k_(off) and K_(D) of the parent anti-ED-A antibody andanti-ED-A antibodies B2, C5, D5, C8, F8, B7 and G9 were evaluated usingSurface Plasmon Resonance. The chip was equilibrated over night with thesame buffer used during the assay at a buffer flow rate of 5 μl/min. Thewhole coating procedure was performed at this flow rate. The antigen11A12 was diluted 1:25 with acetate buffer pH 4.00 (provided byBIACORE™) to a final concentration of 20 μg/ml. The NHS and EDC werethen mixed and 50 μl injected to activate the COOH groups on the CM5chip. This was followed by injection of 40 μl of the antigen (this lastsabout 40″). Then 30 μl of Ethanolammine were injected in order to blockthe reactivity of eventual free COOH.

Each sample was assayed at a flow rate 20 μl/min. 20 μl of undilutedmonomeric protein (as it comes out from the gel filtration) wasinjected. The dissociation time was left to run for about 200″. Then 10μl of HCl 10 mM was injected to regenerate the chip. The injection ofmonomeric protein was repeated at different dilutions, i.e. 1:2 dilution(in PBS) followed by regeneration with HCl. This was followed by a thirdinjection of the protein, at a dilution of 1:4 followed again byregenartion with HCl. The k_(on), k_(off) and KD values for eachanti-ED-A antibody were evaluated using the BIAevaluation software.

Selection of Anti-ED-A Antibodies

BIAcore™ Analysis 1

The BIAcore™ analysis produced a graph for each anti-ED-A antibody whichwas analysed to deduce the affinity of an antibody for the antigen asfollows: The x axis of each graph corresponds to time and the y axiscorresponds to Resonance Units (a measure which indicates the bindingaffinity of the tested antibody for the antigen coated onto the BIAcore™chip). Each graph showed 3 peaks and 1 dip which correspond to changesof buffer and are therefore irrelevant for the interpretation of theresults.

The ascending part of each graph represents the association phase. Thesteeper is the curve in this part of the graph, the faster is theassociation of the antibody with the antigen. The descending part ofeach graph represents the dissociation phase of the antibody from theantigen. The flatter the curve in this part of the graph is, the sloweris the dissociation of the antibody from the antigen.

Anti-ED-A antibodies H1, B2, C5, D5, E5, C8, F8, F1, B7, E8 and G9 allshowed a flatter dissociation curve than the parent anti-ED-A antibodyfrom which they were derived, indicating that they bind ED-A, and hencealso A-FN, with a greater affinity than the parent anti-ED-A antibody.The graphs for antibodies E5, F1, F8 and H1 showed the flattestdissociation curves of all the anti-ED-A antibodies tested. Theassociation curves of antibodies H1, C5, D5, E5, C8, F8 and F1 wereflatter than that observed for the parent anti-ED-A antibody while theassociation curve observed for antibodies B2, B7, E8 and G9 was as steepas the association curve observed for the parent anti-ED-A antibody.However, as bacterial supernatants of IPTG-induced E. coli TG-1 cellswere used for the BIAcore™ analysis of antibodies H1, B2, C5, D5, E5,C8, F8, F1, B7, E8 and G9, the concentration of the tested antibodysamples was unknown but most probably lower than the concentration ofthe parent anti-ED-A antibody sample used for comparison. Consequently,the association curve of antibodies H1, B2, C5, D5, E5, C8, F8, F1, B7,E8 and G9 may be artificially low due to the low concentration ofantibody in the samples used for the BIAcore™ analysis. However, asconcentration does not significantly affect the dissociation of anantibody from its target antigen in BIAcore™ analysis, the flatdissociation curves observed for antibodies H1, B2, C5, D5, E5, C8, F8,F1, B7, E8 and G9 show that these antibodies bind ED-A with at least anequal, and probably a higher affinity, than the parent anti-ED-Aantibody.

BIAcore Analysis 2

The k_(on), k_(off) and KD values for each anti-ED-A antibody wereevaluated using the BIAevaluation software. The k_(on), k_(off) and KDvalues of the parent anti-ED-A antibody and anti-ED-A antibodies B2, C5,D5, C8, F8, B7 and G9 for antigen 11A12 are detailed in Table 2.Anti-ED-A antibodies B2, C5, D5, C8, F8, B7 and G9 all have a betterK_(D) values for antigen 11A12 than the parent anti-ED-A antibody fromwhich they were derived, indicating that they bind ED-A, and hence alsoA-FN, with a greater affinity than the parent anti-ED-A antibody.

Sequences

Anti-ED-A antibodies H1, B2, C5, D5, E5, C8, F8, F1, B7, E8 and G9 areall scFv antibodies and were sequenced using conventional methods. Thenucleotide sequence of the anti-ED-A antibody H1 is shown in FIG. 3. Theamino acid sequence of the anti-ED-A antibody H1 is shown in FIG. 4.

Preferred nucleotide sequences encoding VH and/or VL of anti-ED-Aantibodies B2, C5, D5, E5, C8, F8, F1, B7, E8 and G9 are identical tonucleotide sequences encoding VH and/or VL of anti-ED-A antibody H1,except that the nucleotide sequences encoding the H1 CDR1s of the light(VL) and heavy (VH) chain are substituted with the nucleotide sequencesencoding the light (VL) and heavy (VH) chain CDR1s listed in Table 1 forthe respective antibody.

The preferred nucleotide sequences encoding the VH and/or VL ofanti-ED-A scFv F8 diabody are identical to the nucleotide sequencesencoding VH and/or VL of anti-ED-A antibody H1, except that thenucleotide sequences encoding the H1 CDR1s of the light (VL) and heavy(VH) chain are substituted with the nucleotide sequences encoding thelight (VL) and heavy (VH) chain CDR1s listed in Table 1 for anti-ED-Aantibody F8. The preferred nucleotide sequence encoding the linkerlinking the VH and VL of the anti-ED-A scFv F8 diabody isgggtccagtggcggt (SEQ ID NO: 29).

Anti-ED-A antibodies B2, C5, D5, E5, C8, F8, F1, B7, E8 and G9 haveidentical amino acid sequences to anti-ED-A antibody H1, except that theamino acid sequences of the H1 CDR1s of the light (VL) and heavy (VH)chain are substituted with the amino acid sequences of the light (VL)and heavy (VH) chain CDR1s listed in Table 1 for the respectiveantibody. The amino acid sequence of the anti-ED-A scFv F8 diabody isidentical to the amino acid sequences of anti-ED-A antibody H1, exceptthat the amino acid sequences of the H1 CDR1s of the light (VL) andheavy (VH) chain are substituted with the amino acid sequences of thelight (VL) and heavy (VH) chain CDR1s listed in Table 1 for anti-ED-Aantibody F8, and the amino acid sequence of the linker in H1 issubstituted with the linker amino acid sequence GSSGG (SEQ ID NO: 28).

The amino acid sequence of the anti-ED-A antibody B2 VH domain (SEQ IDNO: 21) is identical to the amino acid sequence of the VH domain ofanti-ED-A antibody H1 except that SEQ ID NO: 23 is substituted for theVH CDR1 of H1.

The amino acid sequence of the anti-ED-A antibody C5 VH domain (SEQ IDNO: 41) is identical to the amino acid sequence of the VH domain ofanti-ED-A antibody H1 except that SEQ ID NO: 43 is substituted for theVH CDR1 of H1.

The amino acid sequence of the anti-ED-A antibody D5 VH domain (SEQ IDNO: 51) is identical to the amino acid sequence of the VH domain ofanti-ED-A antibody H1 except that SEQ ID NO: 53 is substituted for theVH CDR1 of H1.

The amino acid sequence of the anti-ED-A antibody E5 VH domain (SEQ IDNO: 61) is identical to the amino acid sequence of the VH domain ofanti-ED-A antibody H1 except that SEQ ID NO: 63 is substituted for theVH CDR1 of H1.

The amino acid sequence of the anti-ED-A antibody C8 VH domain (SEQ IDNO: 71) is identical to the amino acid sequence of the VH domain ofanti-ED-A antibody H1 except that SEQ ID NO: 73 is substituted for theVH CDR1 of H1.

The amino acid sequence of the anti-ED-A antibody F8 VH domain (SEQ IDNO: 81) is identical to the amino acid sequence of the VH domain ofanti-ED-A antibody H1 except that SEQ ID NO: 83 is substituted for theVH CDR1 of H1. The VH domain of the anti-ED-A F8 diabody has the sameamino acid sequence as VH domain of the anti-ED-A antibody F8 (i.e. SEQID NO: 81).

The amino acid sequence of the anti-ED-A antibody F1 VH domain (SEQ IDNO: 91) is identical to the amino acid sequence of the VH domain ofanti-ED-A antibody H1 except that SEQ ID NO: 93 is substituted for theVH CDR1 of H1.

The amino acid sequence of the anti-ED-A antibody B7 VH domain (SEQ IDNO: 101) is identical to the amino acid sequence of the VH domain ofanti-ED-A antibody H1 except that SEQ ID NO: 103 is substituted for theVH CDR1 of H1.

The amino acid sequence of the anti-ED-A antibody E8 VH domain (SEQ IDNO: 111) is identical to the amino acid sequence of the VH domain ofanti-ED-A antibody H1 except that SEQ ID NO: 113 is substituted for theVH CDR1 of H1.

The amino acid sequence of the anti-ED-A antibody G9 VH domain (SEQ IDNO: 31) is identical to the amino acid sequence of the VH domain ofanti-ED-A antibody H1 except that SEQ ID NO: 33 is substituted for theVH CDR1 of H1.

The amino acid sequence of the anti-ED-A antibody B2 VL domain (SEQ IDNO: 22) is identical to the amino acid sequence of the VL domain ofanti-ED-A antibody H1 except that SEQ ID NO: 26 is substituted for theVL CDR1 of H1.

The amino acid sequence of the anti-ED-A antibody C5 VL domain (SEQ IDNO: 42) is identical to the amino acid sequence of the VL domain ofanti-ED-A antibody H1 except that SEQ ID NO: 46 is substituted for theVL CDR1 of H1.

The amino acid sequence of the anti-ED-A antibody D5 VL domain (SEQ IDNO: 52) is identical to the amino acid sequence of the VL domain ofanti-ED-A antibody H1 except that SEQ ID NO: 56 is substituted for theVL CDR1 of H1.

The amino acid sequence of the anti-ED-A antibody E5 VL domain (SEQ IDNO: 62) is identical to the amino acid sequence of the VL domain ofanti-ED-A antibody H1 except that SEQ ID NO: 66 is substituted for theVL CDR1 of H1.

The amino acid sequence of the anti-ED-A antibody C8 VL domain (SEQ IDNO: 72) is identical to the amino acid sequence of the VL domain ofanti-ED-A antibody H1 except that SEQ ID NO: 76 is substituted for theVL CDR1 of H1.

The amino acid sequence of the anti-ED-A antibody F8 VL domain (SEQ IDNO: 82) is identical to the amino acid sequence of the VL domain ofanti-ED-A antibody H1 except that SEQ ID NO: 86 is substituted for theVL CDR1 of H1. The VL domain of the anti-ED-A F8 diabody has the sameamino acid sequence as VL domain of the anti-ED-A antibody F8 (i.e. SEQID NO: 82).

The amino acid sequence of the anti-ED-A antibody F1 VL domain (SEQ IDNO: 92) is identical to the amino acid sequence of the VL domain ofanti-ED-A antibody H1 except that SEQ ID NO: 96 is substituted for theVL CDR1 of H1.

The amino acid sequence of the anti-ED-A antibody B7 VL domain (SEQ IDNO: 102) is identical to the amino acid sequence of the VL domain ofanti-ED-A antibody H1 except that SEQ ID NO: 106 is substituted for theVL CDR1 of H1.

The amino acid sequence of the anti-ED-A antibody E8 VL domain (SEQ IDNO: 112) is identical to the amino acid sequence of the VL domain ofanti-ED-A antibody H1 except that SEQ ID NO: 116 is substituted for theVL CDR1 of H1.

The amino acid sequence of the anti-ED-A antibody G9 VL domain (SEQ IDNO: 32) is identical to the amino acid sequence of the VL domain ofanti-ED-A antibody H1 except that SEQ ID NO: 36 is substituted for theVL CDR1 of H1.

Optionally, the amino acid at position 5 of the VH domain of anti-ED-Aantibodies H1, B2, C5, D5, E5, C8, F8, F1, B7, E8, G9 and the scFv F8diabody may be a leucine residue (L) rather than a valine residue (V) asshown in FIG. 4A. In addition, or alternatively, the amino acid atposition 18 of the VL domain of anti-ED-A antibodies H1, B2, C5, D5, E5,C8, F8, F1, B7, E8, G9 and the scFv F8 diabody may be an arginineresidue (R) rather than a lysine residue (K) as shown in FIG. 4C.

Cloning, Production and Characterization of F8-IL10

The human IL-10 gene was amplified by PCR using the following primersequences:

a backward antisense primer,5′-TCGGGTAGTAGCTCTTCCGGCTCATCGTCCAGCGGCAGCCCAGGCCAGGGCACC-3′(SEQ ID NO: 144); and a forward sense primer,5′-TTTTCCTTTTGCGGCCGCtcattaGTTTCGTATCTTCATTGTCATGT A-3′(SEQ ID NO: 145),which appended part of a 15 amino acid linker (SSSSG)3 (amino acids243-257 of SEQ ID NO: 149) at its N-terminal and stop codon and NotIrestriction site at its C-terminal.

DNA encoding the single-chain variable fragment (F8) was amplified witha signal peptide using the following primer pairs:

a backward antisense primer, 5′-CCCAAGCTTGTCGACCATGGGCTGGAGCC-3′(SEQ ID NO: 146) and a forward sense primer, 5′-GAGCCGGAAGAGCTACTACCCGATGAGGAAGAGAATTCTTTGATTTCCACCTTGGTCCCTTG-3′(SEQ ID NO: 147).

Using this strategy, a HindIII restriction site was inserted at theN-terminal and a complementary part of the linker sequence was insertedat the C-terminal.

The single-chain Fv and IL-10 fragments were then assembled using PCRand cloned into the HindIII and NotI restriction sites of the mammaliancell-expression vector pcDNA3.1(+).

CHO-S cells were stably transfected with the previously describedplasmids and selection was carried out in the presence of G418 (0.5g/l).

Clones of G418-resistant cells were screened for expression of thefusion protein by ELISA using a recombinant EDA of human fibronectin asantigens and Protein A for detection.

The fusion proteins were purified from the cell-culture medium byaffinity chromatography over Protein A columns.

The size of the fusion proteins was analysed in reducing and nonreducingconditions on SDS-PAGE and in native conditions by FPLC gel filtrationon a Superdex S-200 exclusion column (Amersham Pharmacia Biotech,Dübendorf, Switzerland).

Activity Assay

Biological activity of hIL10 was determined by its ability to induce theIL-4 dependent proliferation of MC/9 cells (Thompson-et al., 1991) usingthe colorimetric MTT dye-reduction assay.

10.000 MC/9 (ATCC, Manassas, USA) cells/well in 200 μl of mediumcontaining 5 pg (0.05 Units) of murine IL4/ml (eBiosciences) in a96-well microtiter plate were treated for 48 hr with varying amounts ofhuman IL10. The hIL10 standard and the F8-IL10 fusion protein were usedat a maximum of 100 ng/ml IL10 equivalents and serially diluted. 10 μlof 5 mg/ml MTT (Sigma) was added and incubated for 3-5 hr. The cellswere then centrifuged, lysed with DMSO and read for absorbance at 570nm.

References

All references cited anywhere in this specification, including thosecited anywhere above, are hereby incorporated by reference in theirentirety and for all purposes.

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TABLE 1 Nucleotide and amino acid sequences of theheavy chain (VH) and light chain (VL) CDR1sof the anti-ED-A affinity matured antibodies Antibody CDR1 (VH)CDR1 (VL) H1 CCG CGG AGG TCT GCG TGG P   R   R S   A   W (SEQ ID NO: 3)(SEQ ID NO: 6) B2 GCG GCT AAG GTG GCT TTT A   A   K V   A   F(SEQ ID NO: 23) (SEQ ID NO: 26) C5 CCG ATT ACT TTG CAT TTT P   I   TL   H   F (SEQ ID NO: 43) (SEQ ID NO: 46) D5 GTG ATG AAG AAT GCT TTTV   M   K N   A   F (SEQ ID NO: 53) (SEQ ID NO: 56) E5 ACT GGT TCTCTT GCG CAT T   G   S L   A   H (SEQ ID NO: 63) (SEQ ID NO: 66) C8CTT CAG ACT CTT CCT TTT L   Q   T L   P   F (SEQ ID NO: 73)(SEQ ID NO: 76) F8 CTG TTT ACG ATG CCG TTT L   F   T M   P   F(SEQ ID NO: 83) (SEQ ID NO: 86) F1 TAG GCG CGT GCG CCT TTT Q(Amber) A RA   P   F (SEQ ID NO: 93) (SEQ ID NO: 96) B7 CAT TTT GAT CTG GCT TTTH   F   D L   A   F (SEQ ID NO: 103) (SEQ ID NO: 106) E8 GAT ATG CATTCG TCT TTT D   M   H S   S   F (SEQ ID NO: 113) (SEQ ID NO: 116) G9CAT ATG CAG ACT GCT TTT H   M   Q T   A   F (SEQ ID NO: 33)(SEQ ID NO: 36)

TABLE 2 BIAcore evaluation data Antibody k_(on) (1/Ms) k_(off) (1/s)K_(D) (M) Parent  2.5 × 10⁵ 0.02   ~1 × 10⁻⁷ anti-ED-A antibody B2  3.8× 10⁵ 7.54 × 10⁻³    ~2 × 10⁻⁸ C5 3.04 × 10⁵ 9.23 × 10⁻³    ~3 × 10⁻⁸ D54.53 × 10⁵ 7.6 × 10⁻³ ~1.7 × 10⁻⁸ C8  3.8 × 10⁵ 5.3 × 10⁻³ ~1.4 × 10⁻⁸F8 4.65 × 10⁵ 1.4 × 10⁻³ ~3.1 × 10⁻⁹ B7 2.67 × 10⁵ 4.5 × 10⁻³ ~1.68 ×10⁻⁸  G9  3.6 × 10⁵ 7.54 × 10⁻³  ~2.09 × 10⁻⁸ 

1. An antibody that binds the ED-A of fibronectin, comprising a VHdomain which has amino acid sequence SEQ ID NO: 81 and a VL domain whichhas amino acid sequence SEQ ID NO: 82, and is conjugated tointerleukin-10 (IL-10).
 2. The antibody of claim 1, wherein the antibodyis a diabody.
 3. The antibody of claim 1, wherein the antibody comprisesa single chain Fv.
 4. The antibody of claim 1, wherein the antibody is asmall immunoprotein (SIP).
 5. An antibody that binds the ED-A offibronectin, comprising: a VH domain comprising a framework and a set ofcomplementarity determining regions HCDR1, HCDR2, and HCDR3; and a VLdomain comprising a framework and a set of complementarity determiningregion LCDR1, LCDR2, and LCDR3, wherein the VH domain comprises theamino acid sequence of SEQ ID NO: 81, and the VL domain comprises theamino acid sequence of SEQ ID NO: 82, and wherein the antibody isconjugated to interleukin-10.